Mass Spectrometry for the Clinical Laboratory [1st Edition] 9780128009925, 9780128008713

Table of contents :
Content:
Front matter,Copyright,List of Contributors,PrefaceEntitled to full textChapter 1 - Mass spectrometry in the clinical laboratory: determining the need and avoiding pitfalls, Pages 1-15, W. Clarke
Chapter 2 - Application specific implementation of mass spectrometry platform in clinical laboratories, Pages 17-35, H. Nair
Chapter 3 - Sample preparation techniques for mass spectrometry in the clinical laboratory, Pages 37-62, J. Stone
Chapter 4 - Validation, quality control, and compliance practice for mass spectrometry assays in the clinical laboratory, Pages 63-76, D.F. Stickle, U. Garg
Chapter 5 - Best practices for routine operation of clinical mass spectrometry assays, Pages 77-107, B. Rappold
Chapter 6 - Toxicology: liquid chromatography mass spectrometry, Pages 109-130, K.L. Lynch
Chapter 7 - Toxicology: GCMS, Pages 131-163, P.B. Kyle
Chapter 8 - Therapeutic drug monitoring using mass spectrometry, Pages 165-179, P.J. Jannetto
Chapter 9 - Vitamin D metabolite quantitation by LC-MS/MS, Pages 181-204, H. Ketha, R.J. Singh
Chapter 10 - Steroid hormones, Pages 205-230, J.C. Cook-Botelho, L.M. Bachmann, D. French
Chapter 11 - Mass spectrometry in the clinical microbiology laboratory, Pages 231-245, I.W. Martin
Chapter 12 - High resolution accurate mass (HRAM) mass spectrometry, Pages 247-259, C.A. Crutchfield, W. Clarke
Chapter 13 - Evolving platforms for clinical mass spectrometry, Pages 261-276, J.Y. Yang, D.A. Herold
Index, Pages 277-288

Citation preview

Mass Spectrometry for the Clinical Laboratory

Edited by

Hari Nair, PhD, DABCC, FACB

Boston Heart Diagnostics, Framingham, MA, United States

William Clarke, PhD, MBA, DABCC

Johns Hopkins School of Medicine, Johns Hopkins University, Baltimore, MD, United States

AMSTERDAM • BOSTON • HEIDELBERG • LONDON NEW YORK • OXFORD • PARIS • SAN DIEGO SAN FRANCISCO • SINGAPORE • SYDNEY • TOKYO Academic Press is an imprint of Elsevier

Academic Press is an imprint of Elsevier 125 London Wall, London EC2Y 5AS, United Kingdom 525 B Street, Suite 1800, San Diego, CA 92101-4495, United States 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom Copyright © 2017 Elsevier Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library ISBN: 978-0-12-800871-3 For information on all Academic Press publications visit our website at https://www.elsevier.com/

Publisher: Mica Haley Acquisition Editor: Tari Broderick Editorial Project Manager: Pat Gonzalez Production Project Manager: Julia Haynes Designer: Maria Inês Cruz Typeset by Thomson Digital

List of Contributors L.M. Bachmann, PhD, DABCC Department of Pathology, Virginia Commonwealth University, Richmond, VA, United States W. Clarke, PhD, MBA, DABCC Johns Hopkins University School of Medicine, Baltimore, MD, United States J.C. Cook-Botelho, PhD Clinical Chemistry Branch, Division of Laboratory Sciences, Centers for Disease Control and Prevention, Atlanta, GA, United States C.A. Crutchfield, PhD Johns Hopkins University School of Medicine, Baltimore, MD, United States D. French, PhD, DABCC, FACB Department of Laboratory Medicine, University of California San Francisco, San Francisco, CA, United States U. Garg, PhD Department of Pathology and Laboratory Medicine, Children’s Mercy Hospital, Kansas City, MO, United States D.A. Herold, MD, PhD Department of Pathology, University of California San Diego, La Jolla; VAMC-San Diego, San Diego, CA, United States P.J. Jannetto, PhD, DABCC, FACB, MT(ASCP) Mayo Clinic, Department of Laboratory Medicine and Pathology, Toxicology and Drug Monitoring Laboratory, Metals Laboratory, Rochester, MN, United States H. Ketha, PhD, NRCC Department of Pathology, University of Michigan Hospital and Health Systems, Ann Arbor, MI, United States P.B. Kyle, PhD, DABCC University of Mississippi Medical Center, Jackson, MS, United States K.L. Lynch, PhD, DABCC, FACB Department of Laboratory Medicine, University of California, San Francisco, CA, United States I.W. Martin, MD Dartmouth-Hitchcock Medical Center, One Medical Center Drive, Lebanon, NH, United States of America H. Nair, PhD, DABCC, FACB Boston Heart Diagnostics, Framingham, MA, United States B. Rappold Essential Testing, LLC, Collinsville, IL, United States R.J. Singh, PhD, DABCC Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, United States

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List of Contributors

D.F. Stickle, PhD Department of Pathology, Jefferson University Hospital, Philadelphia, PA, United States J. Stone, MT(ASCP), PhD, DABCC Center for Advanced Laboratory Medicine, University of California San Diego Health System, San Diego, CA, United States J.Y. Yang, PhD Department of Pathology, University of California San Diego, La Jolla, CA, United States

Preface This book was born out of an idea that a select compilation of the illustrated experiences of a panel of expert practitioners of clinical mass spectrometry might be beneficial to those of us who are considering implementation of the art in our own laboratories perhaps for the first time. I would like to thank my mentors and colleagues from the Department of Lab Medicine at the University of Washington as well as those friends that I get to meet at AACC and other venues for their valuable insights that, in part, helped shape the idea for this book. Personally, I saw this project as an opportunity to learn. I feel extremely lucky to have Bill Clarke as my coeditor and mentor in this pursuit. I am grateful for his kind and effective mentorship, encouragements, collaboration, and vast technical and professional insights. First we listed the topics that we felt might be of interest to most clinical labs and then requested some of the best known practitioners in the field to tell their stories on those topics. For their belief in this project, their willingness to contribute, and for their expertise that is so valuable to the clinical chemistry community. I reserve my utmost gratitude to the authors of this book. This project has been in the works for nearly 3 years. Patience and goodwill gestures from many individuals along the way lit its path. Foremost, I would like to thank my wife Rekha and my boys Shreyas and Shree for their patience and for being my inspiration. I am thankful to Ruthi Breazeale, Sr. VP and Dr. Ernst Scheafer, co-founder and medical director at Boston Heart Diagnostics for their encouragements and accommodation. What a pleasure it has been to work with the incredibly professional and pleasant Team Elsevier! Thank you!! My hope is that this book will add at least a drop to the ever growing number of resources that we as a community will need to perfect the art of implementing mass spectrometry in the clinical lab. Hari Nair, PhD, DABCC, FACB

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CHAPTER

MASS SPECTROMETRY IN THE CLINICAL LABORATORY: DETERMINING THE NEED AND AVOIDING PITFALLS

1 W. Clarke

Johns Hopkins University School of Medicine, Baltimore, MD, United States

1  CLINICAL MASS SPECTROMETRY Historically, the complexity of instrumentation and sample preparation has relegated LC-MS based assays to specialized laboratories with extensive technical expertize. Until recently, applications of MS in the clinical laboratory were limited to gas chromatography (GC)-MS for toxicology confirmation testing and testing for inborn errors of metabolism, some GC-MS applications for steroid analysis in specialty laboratories, and inductively coupled plasma (ICP)-MS for elemental analysis. In most cases, this testing has been restricted to specialized laboratories within a hospital, or to large reference laboratories. However, with the simplification of MS instrumentation and introduction of atmospheric spray ion sources along with the emergence of routine liquid-chromatography tandem MS (LC-MS/MS), MS has become a viable option for routine testing in clinical laboratories.

1.1  BASIC MASS SPECTROMETRY CONCEPTS MS is a powerful analytical technology that can be used to identify unknown organic or inorganic compounds, determine the structure of complex molecules, or quantitate extremely low concentrations of known analytes (down to one part in 1012). For MS-based analysis, molecules must be ionized, or electrically charged, to produce individual ions. Thus, MS analysis requires that the atom or molecule of interest has the ability to be ionized and be present in the gas phase. MS instruments analyze molecules by relating the mass of each molecule to the charge; this identifying characteristic is specific to each molecule and is referred to as the mass-to-charge ratio (m/z). Therefore, if the molecule has a single charge (z = 1), the m/z ratio will be equal to the molecular mass. The analytical power of the mass spectrometer lies in its resolution, or the ability to discern one molecular mass from another. The resolution can be determined by examining the width of an m/z peak or the separation between adjacent peaks; a narrow peak with little overlap indicates greater resolution. For two adjacent peaks of masses m1 and m2, the resolving power is defined as m1/(m1 – m2). The expression (m1 − m2) may also be referred to as ∆m. Higher instrument resolution results in increased mass accuracy and the ability to avoid interference from compounds of similar mass that may also be present

Mass Spectrometry for the Clinical Laboratory. http://dx.doi.org/10.1016/B978-0-12-800871-3.00001-8 Copyright © 2017 Elsevier Inc. All rights reserved.

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CHAPTER 1  MASS SPECTROMETRY IN THE CLINICAL LABORATORY

FIGURE 1.1  Schematic Diagram of Mass Spectrometry (MS)

in the sample. Mass accuracy is defined as the mass difference that can be detected by the analyzer divided by the observed, or true mass. Although there are numerous instrument configurations available, MS system operation can be organized into three main segments: (1) generation of ions; (2) separation of ions based on mass and charge in a mass analyzer; (3) detection of ions and instrument output (Fig. 1.1). Depending upon the type of ionization used, these steps fully or partially occur under vacuum pressure to drive ion movement forward through the instrument.

1.2  COMMON ION SOURCES FOR CLINICAL MASS SPECTROMETRY There are a variety of ion sources available for mass spectrometers. Some of these ion sources are “direct ionization sources,” in which analytes are directly ionized from a surface or from a solution. Other sources, such as atmospheric pressure ionization sources, produce ions from analytes in solution and these are more commonly used in clinical assays due to their compatibility with liquid chromatography. Common atmospheric pressure ion sources include: • electrospray ionization (ESI) • atmospheric pressure chemical ionization (APCI) • atmospheric pressure photoionization (APPI) A summary of the strengths and weaknesses for these sources can be found in Table 1.1.

1.2.1  Electrospray Ionization (ESI) ESI is perhaps the most commonly used ionization technique in clinical MS. It is a sensitive ionization technique for analytes that exist as ions in the LC eluent. In ESI, a solvent spray is formed by the application of a high voltage potential held between a stainless steel capillary and the instrument orifice, coupled with an axial flow of a nebulizing gas (typically nitrogen). Solvent droplets from the spray evaporate in the ion source of the mass spectrometer, releasing ions to the gas phase for analysis in the mass spectrometer. In some ESI sources, heat is used to increase the efficiency of desolvation. While ESI is widely used, it is subject to matrix effects, particularly ion suppression, which must be taken into consideration during method development.

1.2.2  Atmospheric Pressure Chemical Ionization (APCI) APCI uses heat and a nebulization gas to form an aerosol of the eluent from an LC system. In contrast to ESI, ions are not formed in solution or liquid phase. Instead, ions are formed in the gas phase using a corona discharge (high voltage applied to a needle in the source) to ionize solvent molecules and analytes in the aerosol. Ions released to the gas phase are then analyzed by the mass spectrometer. During ionization

1 Clinical mass spectrometry

3

Table 1.1  Overview of Three Ionization Techniques Used in Clinical Mass Spectrometry (MS) Ionization Technique

Advantages

Limitations

ESI

• Sensitive ionization technique for polar • May be more sensitive to matrix effects analytes or ions generated in solution compared to APCI • Has broad applicability for relevant analytes in clinical MS • May yield multiply charged ions, which allows for analysis of larger molecules (i.e., >1000 Da)

APCI

• Typically less sensitive to matrix effects than ESI • May provide better sensitivity for less polar analytes

• Typically only singly charged ions are formed, limiting the effective mass range, • May be unsuitable for thermally labile analytes • May yield less absolute signal relative to ESI

APPI

• Works well with nonpolar analytes • In some cases will ionize analytes that do not ionize by either ESI or APCI.

• Demonstrates limited applicability in clinical MS to date.

APCI, Atmospheric pressure chemical ionization; APPI, atmospheric pressure photoionization; ESI, electrospray ionization.

in the APCI source, some thermal degradation may occur, which can lead to a greater degree of fragmentation in electrospray ionization. For analysis using APCI, the analytes of interest should be heat stable and volatile for best results. APCI is often less susceptible to matrix effects (including ion suppression) as compared to ESI, and may be considered for a wide range of applications, including measurement of nonpolar analytes.

1.2.3  Atmospheric Pressure Photoionization (APPI) APPI is an alternative mechanism to ionize analytes eluting from a chromatography system, although it is much less frequently used than ESI or APCI. In APPI, the solvent is first vaporized in the presence of a nebulizing gas (e.g., nitrogen) and then enters the instrument ion source at atmospheric pressure. Once the aerosol is generated, the mixture of solvent and analyte molecules is exposed to a UV light source that emits photons with energy level that is sufficient to ionize the target molecules, but not high enough to ionize unwanted background molecules. Often, an additive to the LC eluent (commonly toluene) is used to increase ionization efficiency in APPI.

1.3  COMMONLY USED MASS ANALYZERS When coupled to an LC system, the mass spectrometer functions as powerful multiplex detector for chromatography. The analyte of interest is ionized in the source of the mass spectrometer by any of a variety of mechanisms as previously discussed. The ions are then directed to the mass analyzer component of the mass spectrometer, where individual ions are selected according to their m/z. Ions produced from small molecule analytes ( 90% of serum phospholipids. For example, zirconia coated silica particles in the Hybrid-SPE plate bind to the phosphate moiety through a Lewis acid:base functionality [23,24]. Differences between these products in the ability to remove polar lysophospholipids as well as more nonpolar phospholipids, in losses of analyte to nonspecific binding, and in recovery of acidic, neutral, and basic analytes have been described [23]. As with PPT, IS and serum/plasma can be mixed in the plate, followed by addition of the precipitating reagent and further mixing. Vacuum or positive pressure is applied, with retention of precipitated proteins and phospholipids in the plate, and flow through of the filtrate containing analytes to a collection plate. Constraints on the nature of the precipitation reagent and on the ratio of serum to precipitant volumes are specific to each vendor [23,24]. The advantages and disadvantages of PLR are the same as those of PPT—but with a major reduction in matrix effect, improvement in robustness, and increased cost.

3.5  COMMERCIAL MEDIA—SUPPORTED LIQUID EXTRACTION SAMPLE PREPARATION PROTOCOL (SLE OR SALL) SLE or support assisted liquid–liquid extraction (SALL) is a means of immobilizing and therefore greatly facilitating automation of LLE [1]. With SLE the aqueous sample is added to the cartridge or plate and spreads out as small droplets widely dispersed in the bed of finely milled diatomaceous earth particles. When immiscible organic solvent is applied, nonpolar analytes partition from the polar aqueous sample with high efficiency in to the solvent. The technique has applicability to a wide range of analytes and can be effective at removing phospholipids. SLE plates and cartridges are available from a number of vendors, including Agilent, Biotage, Merck-Millipore, Thermo-Fisher, and United Chemical Technologies. Sample, IS and a buffer are mixed. The maximum volumes of sample and aqueous diluent that can be used are dictated by the bed mass of the diatomaceous earth. For example, the Biotage ISOLUTE SLE+ plate with a 400 mg bed allows a maximum load volume of 400 µL [25]. A 1:1 dilution of sample with buffer is recommended, limiting the theoretical maximum sample volume to 200 µL [25]. Higher ratios of sample to buffer may be feasible but recovery experiments are necessary for optimization. The sample:buffer mixture is transferred to the SLE plate or cartridge, low vacuum is applied, and ­unlike SPE, the entire sample is absorbed by the bed. There is a 5 min wait while the sample distributes throughout the SLE bed. Then a volume of water-immiscible organic solvent, determined by the bed

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CHAPTER 3  SAMPLE PREPARATION TECHNIQUES

mass, is used for elution. With the 400 mg bed mass example—two applications of 900 µL would be used—the eluent flows through by gravity feed and analytes partition into the eluent which is captured in tubes or plates [25]. As SLE elution solvents are immiscible with water, evaporation of the eluate and reconstitution in a compatible injection solvent is necessary for analysis with reverse phase LC. The advantages of SLE/SALL are ease of automation, particularly when compared to automation of LLE, effective removal of matrix, applicability to a wide range of analyte polarities, and simplicity compared to SPE [1]. The disadvantages are, like other commercial extraction media, increased cost compared to DIL, PPT, and LLE, requirement for a vacuum manifold and evaporator and limited concentrating capability (e.g., maximum sample volume 200–300 µL, if reconstituted in 100 µL, at most a 2.5–3.0 fold concentration).

3.6  COMMERCIAL MEDIA—SOLID PHASE EXTRACTION SAMPLE PREPARATION PROTOCOL (SPE) SPE, whether manual, automated on a liquid handler or online, is essentially a low resolution chromatographic process. Like LLE, SPE was in wide use for HPLC-UV and GC-MS methods prior to the advent of LC-MS/MS. There is abundant literature on SPE sample preparation for LC-MS/MS as well as application notes and extensive support from SPE media vendors [1,26–29]. SPE chemistries for use with aqueous matrices are categorized as ion-exchange, reverse-phase, HILIC, or mixed-mode. Reverse phase SPE is less selective than mixed-mode SPE or LLE and is primarily useful for removing salts and polar matrix components. Nonpolar wash solutions that would remove neutral interferences from reverse phase SPE will also wash analytes to waste, as the only retention mechanism is adsorption to the stationary reverse-phase. In contrast, mixed-mode SPE becomes highly selective by including an anion or cation exchange moiety in the same bed with the reverse-phase component (nonpolar polymer or C18 bonded to silica). This dual functionality is a powerful tool for removing matrix, because charged analytes can be retained with the ion exchange moiety while matrix is removed from the reverse phase with nonpolar wash solutions. A vacuum or positive pressure manifold for cartridges or plates is necessary to perform SPE. Positive pressure moves fluids through the SPE bed more reliably than does vacuum. SPE plates with a small bed mass and hold-up volume (e.g., Waters µElution plate) can be eluted with 15%, further optimization of sample preparation is advised. A quick graphic impression of between sample variability caused by ME can be seen in the plot of IS peak areas versus injection number. Reviewing this plot with each batch is a powerful tool to confirm that the variability of ME between samples remains acceptable, once the assay is validated and has been moved to production. C62 does not list a minimum or a desirable range for percentage extraction recovery, but the spike before and after extraction experiment is useful to interrogate insufficient recovery from transfer, loading, mixing, wash, and elution steps of complex protocols, such as LLE, SPE, SLE, and PRL or losses from any protocol caused by nonspecific adsorption and transfers between containers. Acceptable S/N at the LLOQ is the true measure of acceptable recovery. Grant and Rappold in their short course at the Mass Spectrometry Applications in the Clinical Laboratory (MSACL) meeting recommend first optimizing for reproducibility between different patient samples, and with that achieved, then increasing recovery if necessary [43].

4.3.2  Qualitative Postcolumn Matrix Effect Experiment The qualitative postcolumn infusion experiment visualizes ME over the time course of the chromatographic run [44]. A semi-quantitative assessment of ME can be made, but the best value is the chronologic characterization. It may be possible to modify Rt of the analyte relative to the pattern of ion suppression for a net increase in S/N and robustness [44]. The design of this experiment is to establish a constant MRM signal for both analytes and ISs by infusing neat standards in solvent postcolumn. Injecting a sample without matrix (e.g., water) will demonstrate a baseline profile for comparison with the deflections from ME seen when extracted biological matrix samples are injected (Fig. 3.2). As with the quantitative spiking ME experiment—the more abnormal the native matrix samples used, the better. The primary challenge with this experiment is adjusting the concentrations and infusion rate of the neat standards so the signals are in the midrange of counts per second (cps). Significant deflections can be missed when the infusion signal is too high and fluctuations from noise/baseline drift can make interpretation difficult when signal is too low. Although the goal is to see no loss of signal at analyte Rt, it is common to see some decrease but with closely paralleled deflections between stable labeled ISs and analytes.

4.3.3  Phospholipid Direct Detection Experiment Building an MS/MS method that detects phospholipids directly is a more straightforward, but less comprehensive means than postcolumn infusion to detect the time course of ion suppression for serum

4 Evaluation of sample preparation protocols

47

FIGURE 3.2  Post Column Infusion to Assess Matrix Effect A schematic of the plumbing for post column infusion is inset. LC flow is from the pumps through the autosampler and column to the MSMS. Neat standard is introduced to the LC stream between the column and the MSMS through a syringe and T connection fitting. Extracted ion chromatograms (XIC) for MRMs detecting 11-Nor-9-Carboxy-delta-9-tertrahydrocannabinol (THC-COOH) and deuterium labeled IS are shown overlaid for an injection of blank injection solvent (no matrix effect) and an injection of an extracted urine sample negative for THC-COOH. The arrow denotes the expected retention time (Rt) for THC-COOH. Ion suppression is seen prior to the THC-COOH Rt in the Pt. 2 XIC, but suppression is minimal and similar for analyte and IS at the THC-COOH Rt. Urine was extracted with mixed-mode strong anion exchange SPE.

samples. Our laboratory has found this to be a complementary technique to postcolumn infusion and easier to integrate into a development workflow. Several options have been described, one popular method uses collision induced dissociation (CID) in the source and MRMs 184/184 m/z and 104/104 m/z to detect fragments common to both late and early eluting phospholipids [18,19,21]. Glycerophospholipids, such as phosphatidyl choline, have a 3-carbon glycerol backbone that may be esterified with two fatty acids and a phosphate group. Lysophospholipids are a subgroup of the glycerophospholipid family with one of the hydroxyl groups on the three carbon glycerol backbone remaining unesterified, that is, containing only one fatty acid and a phosphate group. Predictably, lysophospholipids are relatively polar and an earlier eluting source of ion suppression compared to later eluting glycerophospholipids that have two fatty acid chains. Detecting both classes is informative as changes in LC methods and sample preparation may affect them differently [18,19,21]. Goals for direct phospholipid detection are an MS/MS acquisition that is quick to set up, minimizes duty cycle, and broadly detects both early and late eluting species. Xia and Jemal compared three MS/ MS acquisition modes for detecting phospholipids and found that programing one MRM to represent each of the major phospholipid groups had greater selectivity, producing well-defined, quantifiable

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CHAPTER 3  SAMPLE PREPARATION TECHNIQUES

peaks, as compared to more comprehensive or generic acquisition strategies (e.g., precursor/neutral loss scans or in-source CID with m/z 184/184 and 104/104) [18]. Our experience evaluating PRL media supports this recommendation—we used in positive mode the MRMs m/z 524/184, 496/184 (lysophospholipids) and 704/184, 758/184, 786/184, 806/184 (other glycerophospholipids).

4.3.4  Matrix Mixing Experiment C62 recommends matrix admixing experiments that are described in detail in the CLSI document EP07 [33]. For example, matrix A is mixed with matrix B in the percentage ratios 100:0, 75:25, 50:50, 25:75, and 0:100. If ME is significantly different between the two matrices, observed concentrations will differ from expected. This protocol is appealing because it is simple, but it provides less information that the other three experiments.

4.4  EVALUATING METHOD PERFORMANCE A lack of robustness in sample preparation can be a contributor to imprecision, inaccuracy, nonlinearity, and unacceptable limits of quantitation [1]. Except for S/N at the LLOQ, establishing the extent to which sample preparation causes unacceptable method performance, rather than or in addition to, contributions from suboptimal LC or MS/MS conditions, may not be obvious. Switching from an analog to a stable isotope labeled, coeluting IS usually improves precision. Changing to an IS with mass at least +3 m/z above the analyte m/z to correct nonlinearity from high analyte contributions to IS signal is also a simple fix. The quantitative ME and recovery spiking experiment is more time consuming but is valuable for interrogating sample preparation as a possible source of unacceptable method performance. LLOQ can be improved by concentrating analytes to a greater degree, increasing extraction recoveries, and decreasing ion suppression by optimizing LLE, SLE, and SPE protocols. With a fully optimized method, the variance inherent to manual pipetting of sample and IS may ultimately be the limiting factor for precision.

4.5  EVALUATING PRACTICALITY It is the norm to underestimate the amount of time consumed and potential for error in the repetitive sample sorting, labeling, sealing/unsealing, reracking of extraction/injection containers, and in the transfer of liquids between containers that is inherent to manual sample preparation for LC-MS/MS. Every sorting, racking, transfer, sealing, and labeling step that can be eliminated by creative attention to the process not only reduces labor costs, but also reduces the risk of sample misidentification, ergonomic injuries, lowers consumable costs, and may decrease variance from losses of analyte due to nonspecific adsorption to containers and transfer processes. A reduction in the number of transfers between containers, by pipetting or eluting directly into the injection vial or plate rather than in to an interim container can be very efficient but the compromise may be pipetting of much smaller volumes of sample, IS and elution solvent. Low volume pipetting can increase imprecision and evaporation of small volumes of organic solvents during processing is an additional risk. It is important to assess in a robust way the potential effects on precision and recovery from reductions in pipetting or elution volumes. Tremendous increases in productivity are possible by adoption of 96-well plates for sample preparation instead of tubes/vials [3,4]. However, it is significantly easier to mislocate samples when manually pipetting small, colorless, liquid volumes into 96-well plates instead of tubes. Light boxes and other

4 Evaluation of sample preparation protocols

49

pipetting aids can facilitate manual addition of samples to plates, but using automated liquid handling, when feasible, is the best option. Using automated liquid handlers for LC-MS/MS sample preparation not only improves precision and reduces labor costs, but also saves time spent sorting samples and reduces sample misidentifications through positive identification with barcodes.

4.6  EVALUATING ROBUSTNESS The definition of method robustness varies widely between laboratories. In some settings—a column lifetime of several hundred injections is a desirable trade-off for a faster, simpler sample preparation protocol. In contrast—another laboratory may routinely perform extensive, sometimes automated, sample clean up and define >10,000 injections as the minimum acceptable lifetime for LC columns. In one setting, cleaning of the MS/MS interface (section of the MS/MS under vacuum between the source and the quadrupole rail) on a monthly or even weekly basis is accepted as the unavoidable outcome of high volume testing with minimal sample cleanup. To another laboratory—this additional labor, loss of instrument time, and operational unpredictability would be too problematic. Significant cost and labor would be invested to develop and use in production a sample preparation protocol that removes more matrix. Sufficient sample cleanup that introduces less matrix to the LC-MS/MS can yield an MS/MS interface that requires only scheduled 6 month or annual maintenance and a quadrupole rail that never needs cleaning. Fit for purpose, sample preparation is defined here as a protocol that delivers acceptable method performance and removes sufficient matrix to confer the desired, predictable interval of good LC column and MS/MS performance. Few laboratories, if any, can devote the resources to test two different sample preparation protocols for the same analyte on two different instruments over weeks and months to compare method performance, column lifetime, and MS/MS response as measures of robustness. Therefore, the “measure” of method robustness is usually an impression or a series of anecdotes, rather than quantitative data. Process metrics that may be helpful to quantify robustness include: • Tracking peak areas for a representative IS or LLOQ calibrator. Record peak area(s) by date and evaluate for trends, shifts, time course, and against action limits as an indicator of LC-MS/MS sensitivity. • Number of sample repeats required because of ion suppression (low IS peak area relative to calibrators) or MRM ratio failures or interfering peaks. • Number of run interruptions from over-pressure or leaks. • Number of batch failures from unacceptable QC. • Number of batch failures from unacceptable calibration. • Average number and range of injections/column and guard column. • Length of time after cleaning of the MS/MS interface to the next time that cleaning is needed to restore sensitivity. • Number of tubing, fitting, inline frit, rotor seal, column changes because of leaks or over-pressure from clogging. • Number and duration of delays in turn-around time because of LC-MS/MS instrument down-time. • Negative variance of cost/reportable test, caused by frequent repeats, batch failures, delays, instrument downtime.

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CHAPTER 3  SAMPLE PREPARATION TECHNIQUES

The quality of a sample preparation protocol can influence chromatographic robustness and the amount of time needed for data review, although good LC maintenance and optimization of LC, MS/ MS, and peak integration algorithm parameters play a significant role as well. Metrics to quantify chromatographic robustness include: • • • • •

Number of samples/batch requiring manual peak integration. Number of batches with baseline signal above threshold (e.g., > 500 cps). Number of batches with S/N at the LLOQ ≤ 20. Number of batches with unacceptable resolution for critical peak pairs (Rs ≤ 2.0). Number of batches with average peak width and/or peak asymmetry above threshold (e.g., >10 s peak width). • Number of adjustments required in MS/MS acquisition method and data analysis method windows because of Rt shifts caused by column degradation.

5  COMPARISON OF SAMPLE PREPARATION PROTOCOLS Commonly used sample preparation protocols are characterized in Table 3.1 by relative cost and complexity, capability for concentration of analytes, and matrix removal. A major advantage of LC-MS/MS as a measurement technique are the many options for adjusting LC conditions, MS/MS parameters or the sample preparation protocol to compensate for limitations in one of the other phases of the analysis. Thus the choice of sample preparation protocol or complexity of optimization required should always take into account LC and MS/MS functionality. As limitations in personnel, LC-MS/MS expertise, and operating budget are common to most clinical laboratories implementing LC-MS/MS, the first decision about sample preparation is often a consequence of practicality rather than chemistry—whether or not the simple sample preparation protocols—DIL and PPT—will suffice.

Table 3.1  Simplified Comparison of LC-MS/MS Sample Preparation Types

Sample Preparation Protocol

Analyte Dilution (D) or Concentration (C) Possible

Relative Cost

Relative Matrix Relative Complexity Removal

Dilution (DIL)

D

Low

Simple

Less

Protein precipitation (PPT)

D

Low

Simple

Least

Liquid–liquid extraction (LLE)

D or C

Low

Complex

More

Phospholipid removal (LPR)

D

High

Moderately complex

More, selectivea

Supported liquid extraction (SLE) D or C (moderate)

High

Moderately complex

More

Solid phase extraction (SPE)

D or C

High

Complex

More

Online SPE/Turboflow

D or C

High

Complex

More

a

Only phospholipids are removed, other matrix components are not depleted.

5 Comparison of sample preparation protocols

51

The sensitivity of the MS/MS is a first consideration. Purchasing the most sensitive MS/MS the laboratory can afford has many advantages. Sample preparation protocols, such as DIL, PPT, and PRL that do not concentrate analytes are more likely to achieve the desired LLOQ with a higher end MS/ MS. The performance of a more sensitive MS/MS may take longer to degrade because less matrix is necessarily introduced with each injection. Assessing the feasibility of simple sample preparation must take into account the nature of the analyte(s), sample type, throughput requirements, and required quantitation limits. With exceptions, sample preparation for endogenous analytes, such as serum steroid hormones with picomolar quantitation limits requires complex protocols—LLE, SPE, or SLE—that can concentrate analytes and deplete matrix. In contrast, drugs and metabolites at higher concentrations are often amenable to DIL, PPT, or PRL, even with a less sensitive MS/MS. Therefore, an initial method development task is to define oncolumn detection limits and the LLOQ needed. If the desired LLOQ can be met with simple techniques, sample type is the next parameter to ­consider. DIL is appropriate for low protein matrices, such as urine whereas PPT or PRL are appropriate for high protein matrices, such as serum/plasma/whole blood. PPT with manual pipetting may be a reasonable choice with small sample numbers ( pKa, and for basic compounds, at least 2 units  0.98.

5.3 ACCURACY With any method validation, a comparison of methods experiment is required to estimate the inaccuracy or systematic error. TDM tests in our laboratory are typically verified using a combination of patient comparison samples with a reference laboratory and proficiency testing (PT) samples or commercial standards (certified reference material), if available. Fig. 8.2 shows the method comparison (n = 69) of the busulfan by LC-MS/MS to GC-MS (reference method) along with the difference plot (BlandAltman plot). The results were compared using a standard linear regression. The acceptance criteria were that the mean difference between the results should be < 10% with no individual value > 20% and the slope = 1.0 ± 0.1, 95% confidence interval of the intercept should span 0, and the r2 > 0.98.

5.4  REFERENCE INTERVALS For TDM tests, true reference range studies would be challenging for most laboratories to enroll patients taking the desired drug, collect samples at the appropriate time (peak/trough), and correlate it with clinical outcome (efficacy/toxicity). As a result, published reference ranges from clinical trials, clinical practice guidelines, or other sources are often cited. However, it is important to review how the published reference ranges were established and look at the methodology used, patient population, etc., to see how it compares to the laboratory’s patient population. Where available, the laboratory should still verify the reference interval with local patient samples where clinical history can be obtained. Alternately, some laboratories have used the data collected from the reference laboratory in the method comparison study to calculate or adjust their reference range based on the mathematical relationship determined between the two analytical methods. This approach is dangerous since all MS assays aren’t standardized and large variations can be seen. In the case of tacrolimus, studies have shown how independently calibrated LC-MS/MS assays without traceability to an accepted reference method or standard reference material could generate significantly different results [17].

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CHAPTER 8  THERAPEUTIC DRUG MONITORING

FIGURE 8.2  Method Comparison (n = 69) of Busulfan LC-MS/MS (RapidFire) Assay Versus GC-MS (Reference Assay) and the Difference Plot (Bland-Altman plot)

5 Validation considerations

175

5.5 SENSITIVITY The limit of detection (LOD) and limit of quantitation (LOQ) for each TDM assay must be defined. The LOD is the lowest analyte concentration that can be distinguished from the assay background, while the LOQ is the lowest concentration at which the analyte can be quantitated at defined levels for imprecision and accuracy (bias) [18]. In my laboratory, the LOD has been defined as the lowest concentration tested that has a peak height that is greater than or equal to the average of a blank sample (no analyte) plus three standard deviations (SD) of the blank. The acceptance criterion is that the LOD has to be less than 20% of the LOQ. The LOQ in our laboratory for TDM tests is defined as the lowest concentration where the between-day coefficient of variation is less than or equal to 10% with a signal-to-noise ratio for all MRM transitions greater than 10.

5.6  SPECIFICITY (INTERFERENCES) Interference studies are conducted to determine if other commonly coprescribed, used, or abused medications might interfere with the TDM assay. In our laboratory, we run a standard list of the top 25 most commonly prescribed drugs [19], common drugs of abuse, and any other comedications and medications from the same drug class, which are all spiked at 10 µg/mL. Additionally, serum samples are tested for endogenous interferences from hemolysis, icterus, and lipemia. Serum samples are spiked at three different concentrations (subtherapeutic, therapeutic, and toxic) of drug and tested at five different concentrations of hemoglobin, bilirubin, or intralipid at concentrations up to 1,000, 60, and 2,000 mg/dL, respectively. The acceptance criterion is that all samples must match within 10–20% depending on the analyte.

5.7 CARRY-OVER For TDM assays, the laboratory also assesses carryover. For this experiment, the laboratory spikes drug free matrix at concentrations at least two times the upper end of the AMR. The laboratory extracts and analyzes the spiked sample followed by five separate blank (drug-free) samples. The blank samples must be < 20% of the LOQ for the assay. In the end, the laboratory policy and standard operating procedure still requires a technologist to repeat any patient following a sample that is above the AMR, since the assay is not accurate in that concentration range and the true value may be 10 times or 20 times higher where carryover could occur.

5.8  ION SUPPRESSION/ENHANCEMENT With MS, ion suppression/enhancement is a critical factor that can affect a patient’s result. Sample matrix, coeluting compounds, and cross-talk can all contribute to ion suppression/enhancement so it is important that laboratories characterize MS assays [20]. Ion suppression studies for TDM assays are often done by comparing standards spiked into 10 different matrix pools and compared to the drug spiked into aqueous mobile phase. Three different concentrations that span the AMR are prepared and analyzed in triplicate. The acceptance criteria used by the laboratory is that the average ion suppression/ enhancement should be < 20% for the analytes at all levels.

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CHAPTER 8  THERAPEUTIC DRUG MONITORING

5.9  ADDITIONAL VALIDATION STUDIES For TDM assays, the laboratory also performs matrix studies, prepared sample stability studies, and analyte stability studies. As mentioned previously, it is important to measure a drug in the appropriate matrix (i.e., serum, plasma, whole blood). As a result, the laboratory tests a variety of matrices to determine what can or should be accepted. Drug free matrices (serum-red top, sodium heparin plasma-green top, sodium fluoride/potassium oxalate plasma-gray top, potassium EDTA plasma-purple top, and serum separator tube-gold top) are spiked at concentrations level near the low, middle, and high end of the AMR. Pools are mixed and allowed to stand for at least 1 h at room temperature and then analyzed. The pools are then refrigerated and reanalyzed again the next day. The recoveries from the various matrices are then compared. For analyte stability, the results of freshly prepared pools are split and stored under ambient, refrigerated, and frozen (–20°C) conditions. At day 0, 1, 3, 7, 14, and 28, the samples are analyzed and compared back to day 0. Pools are spiked at concentrations near the low, middle and high end of the AMR. In addition, three freeze/thaw cycles are tested. The average difference for any analyte should be < 10% with no individual value > 20%. Based on the recovery results, one time-period for each storage condition is selected as the laboratory’s specimen acceptance criteria along with a preferred storage/transport condition. Lastly, the laboratory examines the stability of the extracted samples by reinjecting the same plate (samples) at least 24 h after the original run on the same instrument while stored at the same temperature. Sample results are then calculated using the calibrators in the original run and recalculated using the calibrators from the reinjected run. This procedure is only done for internal documentation purposes to determine if samples can be reinjected if the MS has a problem during the analysis. The results must match within 10% otherwise the sample extraction process would have to be repeated on fresh patient samples if the MS had any issues.

6  QUALITY CONTROL/QUALITY ASSURANCE CONSIDERATIONS Once a test is validated and goes live, the laboratory must continue to monitor the tests performance per regulatory guidelines, to minimize, identify, and correct analytical errors, ensure optimal MS and method performance, confirm method robustness, and confirm result quality. Three key postimplementation parameters to monitor are shown subsequently.

6.1  PROFICIENCY TESTING (PT) It is a regulatory requirement that PT be performed for all tests. In the case of newer TDM tests, external PT may not be available from the College of American Pathologists (CAP) or other organizations so an alternate PT testing must be done. While each agency (i.e., CAP) has defined acceptance criteria (i.e., 80% pass rate), each laboratory should look for trends/shifts in the results compared to their peer group mean using the same methodology. For TDM tests, several providers are available, such as CAP and LGC Standards. Our laboratory prefers LGC for some of the new TDM analytes, since more LCMS/MS users participate in that survey compared to CAP (i.e., back in 2014, levetiracetam PT for CAP only had ∼16 laboratories using LC-MS/MS compared to ∼35 laboratories for LGC). Table 8.3 shows the results of the PT for 2014 for levetiracetam in the LGC Standards PT program.

177

6 Quality control/quality assurance considerations

Table 8.3  2014 LGC Standards Levetiracetam Results for the Therapeutic Drugs Proficiency Testing Scheme Month

Sample ID

Number of Laboratories Participating

Assigned Value

Method Mean

Laboratory Result

z Score

January

TM 123

37

18.70

18.7

18.3

−0.24

February

TM 124

30

38.75

39.4

35.6

−0.99

March

TM 125

32

75.80

74.2

87.0

1.55

April

TM 126

34

4.00

3.9

3.9

−0.11

May

TM 127

37

23.50

23.3

23.4

−0.04

June

TM 128

36

41.36

41.4

45.3

1.15

July

TM 129

36

7.50

7.6

6.8

−0.67

August

TM 130

30

55.82

56.0

57.0

0.24

September

TM 131

37

9.36

9.5

9.3

−0.05

October

TM 132

36

32.62

32.4

32.7

0.03

November

TM 133

37

14.20

14.3

12.7

−1.06

6.2  QUALITY CONTROL (QC) Again, regulatory guidelines mandate that QC samples be analyzed to verify sample analysis performance. For TDM assays, we use three levels of QC near medical decision points or at least in the subtherapeutic, therapeutic, and toxic range to span the AMR. QC multirules are then established in which the values must fall within plus/minus two SD. While one QC level may fall outside 2 SD, it must be within 3 SD. If the QC continues to fall outside 2 SD on consecutive runs/days, the run would fail and results would have to be repeated.

6.3  SYSTEM SUITABILITY SAMPLES A nonextracted sample made up of standard/internal standard material in a suitable solvent at a known concentration is made up in a large batch and stored based on the stability study data. This sample is then commonly run before a run to determine if the MS is performing properly. These samples can also be run during a run, or after a run/batch. Our laboratory uses these samples after any maintenance to assess how things are operating. For this sample, the retention time, peak shape, height/width, and analyte/IS ratio is recorded and compared to previous day’s runs. For all TDM MS tests, the technologists also have to systematically review each run after analysis. The technologists first make sure that all peaks are properly integrated and the retention times are within ±0.1 min of the standards. The peak shape and integrity are reviewed to look for shouldering or tailing. If any interference is discovered, the sample must be repeated straight or at an appropriate dilution to try and resolve the interference. For the calibrators, the percentage accuracy for each standard must also fall within 10% and the r2 > 0.98. The internal standard metric plot is also reviewed. The internal standard data is then exported into an Excel spreadsheet where the percentage mean of

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CHAPTER 8  THERAPEUTIC DRUG MONITORING

the internal standard is analyzed and flagged, if it falls outside the limits established during method validation. This allows the technologist to see if any ion enhancement or ion suppression may be affecting a result. Furthermore, the blank must not show any carryover and the dilution control (if diluted patient samples were analyzed) must also pass acceptance criteria. Together, these parameters verify the instrument and sample analysis performance and are an essential component of a postimplementation plan.

7 CONCLUSIONS MS is powerful quantitative technique that can be used for TDM. MS offers clinical laboratories several advantages including its specificity, sensitivity, throughput, and cost-effective testing. However, MS is not without its challenges especially with the lack of standardization for TDM assays and the fear of additional regulations by the FDA on LDTs. However, many clinical laboratories are successfully using MS to perform TDM testing for the analytes discussed in this chapter. In the end, the MS applications are endless and offer clinical laboratories a way to meet the TDM needs of physicians and patients.

REFERENCES [1] Oellerich M, Armstrong VW. The role of therapeutic drug monitoring in individualizing immunosuppressive drug therapy: recent developments. Ther Drug Monit 2006;28(6):720–5. [2] Steimer W. Performance and specificity of monoclonal immunoassays for cyclosporine monitoring: how specific is specific? Clin Chem 1999;45(3):371–81. [3] Taylor PJ, Tai CH, Franklin ME, Pillans PI. The current role of liquid chromatography-tandem mass spectrometry in therapeutic drug monitoring of immunosuppressant and antiretroviral drugs. Clin Biochem 2011;44(1):14–20. [4] Russell JA, Kangarloo SB. Therapeutic drug monitoring of busulfan in transplantation. Curr Pharm Des 2008;14(20):1936–49. [5] Tesfaye H, Branova R, Klapkova E, Prusa R, Janeckova D, Riha P, et al. The importance of therapeutic drug monitoring (TDM) for parenteral busulfan dosing in conditioning regimen for hematopoietic stem cell transplantation (HSCT) in children. Ann Transplant 2014;19:214–24. [6] Napoli KL. Is microparticle enzyme-linked immunoassay (MEIA) reliable for use in tacrolimus TDM? Comparison of MEIA to liquid chromatography with mass spectrometric detection using longitudinal trough samples from transplant recipients. Ther Drug Monit 2006;28(4):491–504. [7] Warnke C, Meyer zu Horste G, Hartung HP, Stuve O, Kieseier BC. Review of teriflunomide and its potential in the treatment of multiple sclerosis. Neuropsychiatr Dis Treat 2009;5:333–40. [8] Fermiano M, Bergsbaken J, Kolesar JM. Glucarpidase for the management of elevated methotrexate levels in patients with impaired renal function. Am J Health Syst Pharm 2014;71(10):793–8. [9] Voraxaze package insert. Available from: http://www.accessdata.fda.gov/drugsatfda_docs/label/2012/ 125327lbl.pdf; 2012 [10] Zhang Q, Simpson J, Aboleneen HI. A specific method for the measurement of tacrolimus in human whole blood by liquid chromatography/tandem mass spectrometry. Ther Drug Monit 1997;19(4):470–6. [11] Karppi J, Akerman KK, Parviainen M. Suitability of collection tubes with separator gels for collecting and storing blood samples for therapeutic drug monitoring (TDM). Clin Chem Lab Med 2000;38(4):313–20. [12] Koch TR, Platoff G. Suitability of collection tubes with separator gels for therapeutic drug monitoring. Ther Drug Monit 1990;12(3):277–80.

REFERENCES

179

[13] Taylor PJ. Internal standard selection for immunosuppressant drugs measured by high-performance liquid chromatography tandem mass spectrometry. Ther Drug Monit 2007;29(1):131–2. [14] Owen LJ, Keevil BG. Testosterone measurement by liquid chromatography tandem mass spectrometry: the importance of internal standard choice. Ann Clin Biochem 2012;49(Pt 6):600–2. [15] Zheng N, Jiang H, Zeng J. Current advances and strategies towards fully automated sample preparation for regulated LC-MS/MS bioanalysis. Bioanalysis 2014;6(18):2441–59. [16] Clarke WHA, Molinaro RJ, Iyer B, Bachmann LM, Kulasingam V, Bothelo JC, Mason DS, Cao Z, Rappold B, French D, Tacker DH, Garg S, Truscott DH, Gawoski JM, Yu CY, Grant RP, Zhu Y. C62-A liquid chromatography-mass spectrometry methods; approved guideline. Wayne, PA: Clinical and Laboratory Standards Institute; 2014. [17] Levine DM, Maine GT, Armbruster DA, Mussell C, Buchholz C, O’Connor G, et al. The need for standardization of tacrolimus assays. Clin Chem 2011;57(12):1739–47. [18] Armbruster DA, Pry T. Limit of blank, limit of detection and limit of quantitation. Clin Biochem Rev 2008;29(Suppl. 1):S49–52. [19] Bartholomew M. Top 200 Drugs of 2012. Available from: http://www.pharmacytimes.com/publications/issue/2013/July2013/Top-200-Drugs-of-2012; 2013. [20] Annesley TM. Ion suppression in mass spectrometry. Clin Chem 2003;49(7):1041–4.

CHAPTER

VITAMIN D METABOLITE QUANTITATION BY LC-MS/MS

9 H. Ketha*, R.J. Singh**

*Department of Pathology, University of Michigan Hospital and Health Systems, Ann Arbor, MI, United States; **Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, United States

1  PHYSIOLOGICAL ROLE OF VITAMIN D Rickets as a pathological entity has been known since the archaic times but the first scientific description of rickets dates back to the late 1600s [1]. It was not until the early 1920s that findings demonstrating that rickets could be cured with “accessory” food nutrients or by exposing affected children to ultraviolet (UV) light were published [2]. These groups of accessory food nutrients also shown at that time, to cure scurvy by feeding fresh vegetables to sea-faring sailors [3] were termed as vital amines or vitamins by Casimir Funk [4]. McCollum [5] and Edward Mellanby [6] classified vitamins into water or fat soluble compounds [7] and showed that cod liver oil contained the antirachitic compound that could cure experimental dogs made to acquire rickets by restricting sunlight exposure [5,8]. This fat-soluble antirachitic substance, shown to be different than vitamin A by McCollum [5], was named as vitamin D. Orr in 1923 demonstrated that vitamin D stimulates intestinal absorption of calcium [9], whereas Shipley and Holtrop’s work in 1926 showed that bone mineralization is promoted by an increased plasma calcium and phosphate concentrations directly regulated by vitamin D [10,11]. The findings that vitamin D has to be metabolically activated to exert its calcemic effects and that it also plays a rather counter-intuitive role on calcium resorption from bone in presence of parathyroid hormone (PTH) were established during 1950–70 [1,12–14]. In the decades that followed the scientific literature witnessed an exponential growth in the understanding of vitamin D biochemistry along with the advent of advanced quantitative tools including radio immunoassays (RIAs), high pressure liquid chromatography (HPLC) and mass spectrometry (MS) that helped isolate and quantify vitamin D and its metabolites with high degree of accuracy. Vitamin D has been classified as vitamin D3 or D2 based on the source of the vitamin. Vitamin D3 is the mammalian form also called cholecalciferol whereas vitamin D2, also called ergocalciferol, is the plant derived form of vitamin D. In this chapter, the subscripts will be used to designate each form when the distinction is necessary otherwise the metabolite will denote the total (−D3 + −D2) metabolite concentration. Both vitamin D3 and vitamin D2 undergo similar metabolic processes in humans. The vitamin D endocrine system and PTH are the principle regulators of calcium homeostasis in humans [15–17]. The biochemical pathway of vitamin D action has been shown in Fig. 9.1. Vitamin D3 is synthesized from its precursor 7-dehydrocholesterol following a UV-B catalyzed photo isomerization. Vitamin D3 formed in the skin or vitamin D2 obtained from diet is converted to 25-hydroxyvitamin D3 or Mass Spectrometry for the Clinical Laboratory. http://dx.doi.org/10.1016/B978-0-12-800871-3.00009-2 Copyright © 2017 Elsevier Inc. All rights reserved.

181

182

CHAPTER 9  Vitamin D metabolite quantitation by LC-MS/MS

FIGURE 9.1  Vitamin D Metabolic Pathway

2 Evolution of assays for vitamin D metabolites

183

25-hydroxyvitamin D2, the most abundant circulating form by vitamin D 25-hydroxylase also termed as cytochrome P450 (CYP) 2R1 in the liver. Vitamin D3 and vitamin D2 are transported to the liver by the vitamin D binding protein (VBP). VBP has a high affinity for vitamin D and its metabolites. Majority of the circulating 25(OH)D is bound to VBP in circulation [18]. PTH controls physiological calcium demand by regulating the conversion of 25(OH)D to the active hormone, 1,25-dihydroxyvitamin D (1,25(OH)2D) by inducing the action of 25-hydroxyvitamin D3-1-α-hydroxylase (CYP27B1). 24-hydroxylation of 1,25(OH)2D by 25-hydroxyvitamin D3-24-hydroxylase (CYP24A1) produces a trihydroxylated metabolite 1,24,25(OH)3D which is finally excreted as calcitroic acid in urine [19]. Therefore, CYP24A1 deactivates the active metabolite of the vitamin D pathway. CYP24A1 also converts 25(OH)D to 24,25-dihydroxyvitamin D (24,25(OH)2D), another inactive vitamin D metabolite [20]. The calcium regulatory effects of vitamin D metabolic pathway occur in conjunction with PTH. PTH functions in a seemingly paradoxical manner in the bone microenvironment where it stimulates bone resorption or bone formation, depending on its concentration and on the duration of exposure. Rapid calcium homeostasis is achieved by PTH by bone resorption whereas its long term effects function to achieve extreme systemic needs and maintain skeletal homeostasis [21]. The action of 1,25(OH)2D is tightly regulated by several factors. This tight feedback regulates the amount of active calcemic hormone present in circulation and provides “protection” from excess 25(OH)D when present. For example, following prolonged sun exposure or high dose vitamin D supplementation circulating 25(OH) D levels increase significantly, but 1,25(OH)2D levels do not change significantly and normal healthy adults do not experience hypercalcemia due to this tight feedback control on calcium homeostasis by PTH and vitamin D metabolism. The biochemistry and utility of quantifying clinically relevant vitamin D metabolites are discussed in later sections.

2  EVOLUTION OF ASSAYS FOR VITAMIN D METABOLITES The vitamin D metabolites that are clinically relevant in the differential diagnosis of disorders of calcium homeostasis include 25(OH)D, C3-epi-25(OH)D, 1,25(OH)2D, and 24,25(OH)2D (Table 9.1). VBP is not commonly measured in clinical laboratories but its role in regulating the bioavailable 25(OH)D (fraction of 25(OH)D not bound to VBP) has caused an increased interest in measuring VBP [22,23]. Quantitative LC-MS/MS methods for VBP have been developed but will not be discussed here [22]. In the early 2000s, several clinical laboratories across United States, Europe, and Australia reported an enormous increase in testing related to vitamin D deficiency [27]. In Australia, an increase from 23,000 tests per year to about 2.5 million tests was reported from 2005 to 2010 [28]. An analysis of the trends in laboratory test volumes for Medicare Part B reimbursements in the United States from 2000 to 2010, showed that the number of vitamin D tests reimbursed per 10,000 tests had increased from less than a 100 in 2000 to a greater than 1500—an increase in reimbursement rate of 15-fold over 10 years [29]. The exponential increase in vitamin D testing is partly due to a revived public interest in vitamin D nutritional status following reports showing wide prevalence of vitamin D deficiency [30,31] along with the numerous studies exploring the physiological role of vitamin D and its metabolites in health and disease [32]. With the growing public demand for vitamin D testing and ensuing increasing testing volume, the interest in vitamin D assays grew and so did the number of assays available for routine use. Two methodologies that have received considerable attention for vitamin D metabolite quantitation are immunoassays and liquid chromatography mass spectrometry (LC-MS/MS).

184

CHAPTER 9  Vitamin D metabolite quantitation by LC-MS/MS

Table 9.1  Clinical Utility of Vitamin D Metabolite Quantitation and Biochemical Effect on PTH and Serum Calcium in Various Pathologic States Vitamin D Metabolite

Reference Interval [21]

Used in the Differential Diagnosis of

25(OH)D

10–65 ng/mL [21] 25–162 nmol/L [21]

• Nutritional Rickets

↓

↑

↓

• Vitamin D deficiency

↓

↑

↓a

↑ Variable

↓ Normal

↑ Normal

↓

↑

↓

• VDDR type II; (1,25(OH)2D is low

Normal

↑

↓

• CYP24A1 mutations; has to be measured in conjunction with 24,25(OH)2D.

Normal

↓

↑

C3-epi-25(OH)D Variable percentage; • To accurately determine the dependent on “native” 25(OH)D present 25(OH)D [24,25] since the two forms may have differential downstream calcemic effects

Variable

Variable

Variable

↑

↑

↓

↑ or normal

↓

↑

↑

↓

↑

• Hypocalcemia in end stage renal disease

↓

↑

↓

• CYP24A1 mutations; has to be measured in conjunction with 24,25(OH)2D; elevated 1,25(OH)2D, recurrant hypercalcemia and kidney stones are common

↑

↓

↑

• Vitamin D Toxicity • Monitoring vitamin D supplementation • VDDR type I

1,25(OH)2D

15–60 ng/mL [21] 36–144 nmol/L [21]

• Vitamin D deficiency • Iatrogenic vitamin D toxicity • Hypercalcemia due to malignancy, primary hyperparathyroidism, recurrant kidney stones, hypercalciuria

24,25(OH)2D and 25(OH) (25(OH) D/24,25(OH)2D)

7–35 [26]

Effect on Vitamin D Metabolitea PTH

a

Serum Calcium

a

Refers to the change in vitamin D metabolite.

Majority of circulating (85%) of 25(OH)D is bound to VBP, a small portion (15%) bound to albumin, and only ∼0.03% circulates as free form [18]. For the assay methodology to quantitate 25(OH)D to be successful, vitamin D and its metabolites need to be dissociated from VBP. The very first assays for 25(OH)D quantitation introduced in the 1970s were based on competitive binding with VBP. The sample preparation involved organic solvent extraction (allowing separation of VBP from vitamin D metabolites) followed by chromatographic separation of 25(OH)D from the dihydroxylated vitamin D metabolites and quantitation

2 Evolution of assays for vitamin D metabolites

185

using a competitive binding assay with VBP [20,33]. With the development of an antibody against 25(OH) D in the 1980s a RIA was developed [34,35]. The early extraction assays were largely manual with tedious solvent extraction steps followed by labor intensive quantitation using a radiolabeled tracer. Turn-around time and throughput demands placed on the clinical laboratories led to immunoassays that were eventually fully automated to meet the clinical and operational needs of the institutions [36]. Automated immunoassays are a mainstay in clinical laboratories as these provide an optimum balance between specificity, sensitivity, and throughput for a variety of analytes. However, lack of specificity and wider intramethod variability especially for steroid quantitation using immunoassays is well known [36–38]. Over 40 metabolites of vitamin D have been reported to date [27,39]. Majority of these are not clinically useful as most have a very short half-life in circulation. However, from an analytical stand point, the presence of a large number of structurally related metabolites can be challenging due to potential interferences and cross reactivity. The nonextraction assays lack a “wash” or a separation step that removes structurally similar analogs or metabolites effectively before the quantitation step. However, nonextraction immunoassay methods may be susceptible to interferences and matrix effects especially due to the lipophilic nature of 25(OH)D and its high affinity to VBP [40]. Significant cross reactivity with 24,25-(OH)2D3, 25,26-(OH)2D3, and 25(OH)D3-26,23-lactone has been observed in several immunoassays [34]. While clinical significance of this interference has been debated, 24,25(OH)2D circulates at about 7–35% of the 25OHD concentration [26] and its presence could potentially cause a misclassification of a patient as vitamin D sufficient. Additionally, commercially available immunoassays may differ greatly in their ability to measure 25(OH)D2 [41,42]. Today several manual and automated immunoassays for 25(OH) are available. Many are produced in a kit format that can be used in a manual or automated platforms [34]. Wide variability in 25(OH)D values of the same sample measured in different laboratories has been shown [41,43]. The mean concentration of the same sample in one study measured by different immunoassay methods ranged from 17 to 36 ng/mL [40]. In view of substantial between-method variability in 25(OH)D values, efforts to standardize 25(OH)D assays have been undertaken. National Institute of Standards and Technology (NIST) standard reference material for vitamin D metabolites is now available [44]. Vitamin D external quality assessment scheme (DEQAS) was formed in 1989 with a goal to achieve and maintain reliability in measurement of 25(OH)D and 1,25(OH)2D by assays in use by clinical laboratories. Clinical laboratories accredited by College of American Pathologists can also use DEQAS as their primary proficiency testing for 25-OHD assays. The April, 2016 DEQAS reports for 25(OH)D shows 918 users performing 25(OH)D quantitation of quality-assurance samples by 28 different methods including LC-MS/MS (Table 9.2). Immunoassay methods are the predominant 81% whereas HPLC and LC-MS/MS combined constitute 19% of the users. Of note there has been an increase in LC-MS/MS users since 2012, when DEQAS reported 11% LC-MS/MS users [34]. Matrix effect refers to the impact of the sample components (e.g., proteins, salts, steroids, drugs, phospholipids, antibodies) other than the analyte of choice on the specificity and sensitivity of an analytical method. Matrix effects pose a significant challenge in immunoassays as they can lead to falsely elevated or low results [45]. Difference in the calibrator matrix and patient sample matrix, presence of high affinity binding proteins, coextraction of lipophilic components like phospholipids in the sample preparation step are all potential causes of matrix effects in immunoassays. In case of 25(OH)D assays, many immunoassays use a denaturing buffer to release 25(OH)D and other highly lipophilic vitamin D metabolites from VBP. The presence of other lipophilic components in the patient sample make the complete dissociation and capture of 25(OH)D difficult and nonspecific interaction of other vitamin D metabolites has proven to be problematic. Needless to say the effectiveness of the denaturing buffer will have a strong impact on assay specificity [46,47].

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CHAPTER 9  Vitamin D metabolite quantitation by LC-MS/MS

Table 9.2  DEQAS 2016 Report Showing 25(OH)D Method, the Number of Participating Laboratories, Mean of Reported Value by the Method Group, Standard Deviation (SD) in the Group and Percent Coefficient of Variation with a Methods Group (%CV) Method

Number of Laboratories

Method Mean nmol/L

SD

%CV

All methods

918

33.6

5.1

15.1

Abbott Architect—New kit (5P02)

42

29.9

2.1

7.0

Abbott Architect—Old kit (3L52)

38

31.9

3.2

10.2

Automate IDS EIA

5

35.4

3.0

8.4

Beckman Access2 Total 25OHD

4

37.8

16.1

42.6

Beckman Unicel DXi Total 25OHD

31

33.7

4.6

13.7

bioMerieux 25OH Vitamin D Total

2

28.3

2.9

10.4

DiaSorin Liaison Total

237

30.4

3.0

9.9

DiaSorin RIA

2

33.8

4.8

14.3

DiaSource 25OH VitD Chem Analyzers

2

33.6

4.3

12.8

Diazyme 25OH VitD EIA

2

39.3

0.6

1.5

Euroimmun ELISA

11

32.2

12.4

38.6

Fujirebio Lumipulse D 25OH Vit D

3

35.1

3.5

10.0

HPLC

23

29.4

1.0

3.4

IDS EIA

17

36.8

6.4

17.9

IDS RIA

6

41.8

4.2

11.4

IDS-iSYS

88

35.7

5.1

12.2

LC-MS/MS

156

34.3

5.4

15.0

Ortho Total 25OHD

5

32.3

4.1

12.0

Roche Total 25OHD

160

34.9

5.4

16.7

Siemens Advia Centaur

65

40.5

4.4

12.5

Tosoh AIA

5

40.7

4.7

11.4

Others

9

32.0–54.5

0–12.4

0–5.3

All reported values represent total 25(OH)D = 25(OH)D2 + 25(OH)D3

Quantitation of 1,25(OH)2D is particularly challenging mainly due to very low (1/1000th of 25(OH)D) circulating concentration. Similar to the earliest methods used, even with contemporary LC-MS/MS methods, appropriate sample preparation methods are crucial to quantitate 1,25(OH)2D accurately. One of the first methods used 20 mL of plasma sample, which was extracted with methanol–chloroform followed by purification by liquid chromatography [48]. Quantitation was achieved by a protein binding assay using chick intestinal VDR. Then assays utilizing the calf thymus VDR,

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which was more stable compared to the chick intestinal VDR became available [49]. This assay was tedious to perform but was reliable and with a number of modifications made to the method over several years, was used widely [50]. The first RIA for 1,25(OH)2D was introduced in 1978. The rabbit antibody used was nonspecific and showed cross reactivity to 25(OH). Due to poor specificity of the antibody, sample extraction and manipulation was very tedious and laborious. Then a RIA that used a simple acetonitrile extraction and a 125I-labeled tracer that allowed gamma counting of the assay was introduced [51]. In one such method, use of sodium periodate treatment of the sample extract leading to the conversion of 24,25(OH)2D3 and 25,26(OH)2D3 to aldehyde and ketone forms greatly enhanced specificity. Immunodiagnostic Systems (Boldon, United Kingdom) developed a immunoenrichment based sample purification method that resulted in enhanced specificity towards 1,25(OH)2D. The delipidated serum samples are treated with an antibody bound to a miniimmunocapsule. The antibody-bound 1,25(OH)2D is eluted, eluant evaporated to dryness, then reconstituted prior to RIA quantitation [52]. Interferences in the Diasorin and IDS assays from 1,25(OH)2D3 26,23-lactone, 1,24,25(OH)3D3, and 1,25,26(OH)3D3 has been demonstrated [35,51,53]. As obvious, 1,25(OH)2D quantitation by immunoassay based methods have been problematic and laborious. The IDS’s immunoenrichment based sample preparation approach has been coupled with LC-MS/MS to achieve highly sensitive and specific quantitation of 1,25(OH)2D [54]. 1,25(OH)2D measurement of qualityassurance samples has been shown in Table 9.3. The April, 2016 DEQAS reports for 1,25(OH)D shows 171 users performing quantitation of quality-assurance samples by nine different methods including LC-MS/MS (Table 9.3). Immunoassay methods are the predominant 92% whereas LC-MS/ MS constitute 8% (compared to 3% in 2012) of the users.

Table 9.3  DEQAS 2016 Report Showing 1,25(OH)2D Method, the Number of Participating Laboratories, Mean of Reported Value by the Method Group, Standard Deviation (SD) in the Group and Percent Coefficient of Variation with a Methods Group (%CV) Method

Number of Laboratories

Method Mean pg/ mL

SD

%CV

All methods

171

96.8

19.4

20.1

AMP RIA

2

71.1

27.6

38.9

Cusabio ELISA

1

50.4

0

0.0

DiaSorin Liaison XL

87

104.7

13.1

12.5

DiaSorin RIA

4

80.9

9.3

11.4

DIASource CT

3

84.9

3.8

4.5

IDS EIA

10

92.4

16.0

17.3

IDS RIA

18

102.7

15.0

14.6

IDS-iSYS

32

76.3

21.9

28.6

LC-MS/MS

14

99.8

24.5

24.5

All reported values represent total 1,25(OH)2D = 1,25(OH)2D2 + 1,25(OH)2D3

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CHAPTER 9  Vitamin D metabolite quantitation by LC-MS/MS

3  CLINICAL UTILITY AND QUANTITATION OF VITAMIN D AND ITS METABOLITES BY LC-MS/MS Accurate quantitation of 25(OH)D and 1,25(OH)2D using nonextraction immunoassays has proven to be challenging as discussed in detail in the previous section. The limitations of immunoassays for accurate quantitation of vitamin D metabolites have been the motivators for clinical laboratories for adopting LC-MS/MS methods for vitamin D metabolite quantitation. In this section, we will discuss the key clinical, technical, and strategic considerations involved in the implementation of mass spectrometry for quantitation of vitamin D and its clinically relevant metabolites. Performance characteristics of LC-MS/MS assays developed by various groups for vitamin D metabolites has been shown in Table 9.4.

3.1  VITAMIN D3 AND VITAMIN D2 3.1.1  Clinical Utility

Vitamin D3 (or vitamin D2), present in food, is absorbed via the intestinal lymphatics [1] where the vitamin exists in the chylomicron fraction. Approximately half of absorbed vitamin D in chylomicrons is transferred to VBP in blood. VBP is the principle chaperone protein that facilitates uptake of vitamin D by the liver while albumin also plays a minor role in transporting vitamin D to the liver for further metabolic conversion. Circulating serum concentration of vitamin D3 or vitamin D2 is not clinically relevant as they are converted rapidly to their 25-hydroxylated metabolite in the liver. The negative feedback of 25(OH)D on the activity of CYP2R1 and is not influenced by plasma calcium and phosphorus concentrations [16]. Table 9.4  Assay Performance Parameters for LC-MS/MS Assays for Clinically Useful Vitamin D Metabolites Assay Parameter

25(OH)D [24,55–61]

1,25(OH)2D [54,62]

24,25(OH)2D [26,54,63,64]

25(OH)D2

25(OH)D3

1,25(OH)2D2

1,25(OH)2D3

24,25(OH)2D2

24,25(OH)2D3

Inter assay imprecision

5.0 16%

2.5–12%

11–13%

6-8%

3.1–10.1%

5.2–7.4%

Intra assay imprecision

4.5–11%

5.7–8.0%

9–11%

5.6–11

3.1–6.2%

7–9%

AMR

2.4–123 ng/ mL

0.4–120 ng/ mL

5.15–206 pg/ mL

4.6–185 pg/ mL

0.5–25 ng/mL

0.1–25 ng/mL

Mean recovery

89–110%

86–108%

112–110%

90–120%

94–100%

90–94%

LOD

0.3–2 ng/mL

0.3–2 ng/mL

2.0 pg/mL

2.7 pg/mL

0.5 ng/mL

0.03 ng/mL

The values are a compilation from several different laboratories. Multiply by 2.5 to convert 25(OH)D3 from ng/mL to nmol/L, by 2.41 to convert 25(OH)D2 from ng/mL to nmol/L, by 2.40 to convert 1,25(OH)2D3 from pg/mL to pmol/L, by 2.33 to convert 1,25(OH)2D2 from pg/mL to pmol/L, by 2.40 to convert 24,25(OH)2D3 from ng/mL to nmol/L, by 2.33 to convert 24,25(OH)2D2 from ng/mL nmol/L.

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3.1.2  Assay Procedure LC-MS/MS methods have been focused at determining concentration of vitamin D3 and/or vitamin D2 in food matrices [65]. Quantitative LC-MS/MS methods used for vitamin D3 and vitamin D2 and their metabolites have many common steps particularly those involved in sample preparation and chromatographic separation. Most sample preparation methods described for 25(OH)D in this chapter will be amenable to vitamin D3 and vitamin D2 quantitation.

3.2  25-HYDROXYVITAMIN D 3.2.1  Biochemistry and Clinical Utility Vitamin D3, is converted to 25(OH)D in the liver [66]. The enzyme, CYP2R1 that catalyzes this reaction has been detected in liver microsomes and liver mitochondria. Supplementation with large doses of vitamin D3 or vitamin D2 results in the parallel increase in circulating levels of total 25(OH)D (25(OH)D3 + 25(OH)D2). Since 25-hydroxylation is a poorly regulated process, the circulating level of 25(OH)D is a very good index of vitamin D reserve. On the contrary 1,25(OH)2D production is highly regulated via CYP27B1 and does not provide information on the vitamin D nutritional status. 25(OH)D concentrations are not affected by phosphate but are affected by induction of CYP24A1, the enzyme responsible for degradation of 25(OH) and 1,25(OH)2D. 25(OH)D quantitation alone is sufficient to identify underlying vitamin D deficiency in a patient. The half-life of 25(OH)D is approximately 1 month in humans [67]. 25(OH)D circulates bound to vitamin D binding protein (VBP) with total concentration in a healthy adult in the 5–80 ng/mL range [21]. At 25(OH)D concentrations above 200 ng/mL cause severe hypercalcemia and comorbidities associated with hypercalcemia including irritability, vomiting, and nephrocalcenosis can develop [68–70].

3.2.2  Assay Procedure Several methods for 25(OH)D quantitation have been reported [55–60,71–73]. The sample preparation steps commonly used in clinical laboratories for LC-MS/MS quantitation of vitamin D and its metabolites are shown in Fig. 9.2. The key factors for achieving optimal sample clean-up for accu­ rately quantitating total 25(OH)D are: (1) 25(OH)D has to be dissociated from VBP, (2) and other serum proteins, (3) interfering vitamin D metabolites and other phospholipids have to be chromatographically separated. Since 25(OH)D3 differs from 25(OH)D2 by 12 Da, the selectivity offered by the quadrupole for specific ion-transitions is sufficient to adequately separate them during MS analysis. The C3-epi25(OH)D quantitation has been described in a later section. Dihydroxylated metabolites (1,25(OH)2D and 24,25(OH)2D) can be easily separated from 25(OH)D chromatographically whose retention times on reverse phase HPLC columns are considerably shorter than 25(OH)D. Sample volume of 100–200 µL has been used to achieve optimal sensitivity. Deproteinization can be achieved by acetonitrile, acetonitrile/sodium hydroxide mixture, methanol or methanol:propanol mixture followed by solid phase extraction (SPE) with C8 or C18 solid phases [57,59–61]. A combination of deproteinization and extraction can be achieved by liquid–liquid extraction (LLE) with hexane or n-heptane [55,56]. An online SPE method (TurboFlow HPLC, formerly Cohesive Technologies, Thermo Fisher Scientific Group, Waltham, Massacheusetts, United States of America) has been used by several clinical laboratories including ours, to achieve an automated sample preparation that can combine sample clean-up and analytical separation [57,60]. TurboFlow technology uses a combination of diffusion, column chemistry, and size exclusion to accomplish online sample clean-up of complex

190

The values shown with the analytes represent normal physiological concentrations. The circulating concentration in normal adults has been shown above the arrows. A PTAD based approach using differential mass tagging approach for 25(OH)D has been described in Fig. 9.5.

CHAPTER 9  Vitamin D metabolite quantitation by LC-MS/MS

FIGURE 9.2  Sample Preparation Steps Commonly Used in Clinical Laboratories for LC-MS/MS Quantitation of Vitamin D and its Metabolites

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matrixes prior to HPLC or UHPLC separation, while improving selectivity for the analyte of choice. Online SPE involves injecting/loading the sample extract either directly from a LLE or SPE extraction or after drying/reconstitution from SPE or LLE step onto a C8, C18 or an equivalent cartridge or a TurboFlow column wherein higher flow rates can be facilitated allowing the matrix components to be effectively washed in a weakly organic mobile (loading) phase while retaining the analyte of choice. The sample is then transferred onto an analytical column followed by elution achieved by an increasing organic phase gradient (elution phase). Similar systems like 2-dimensional HPLC, capable of achieving comparable functionalities are offered by other vendors. Increased throughput can be achieved by a multiplexed HPLC or UPLC systems. One example of a multiplex HPLC system is the TurboFlow TLX4 HPLC system (now Transcend II System with Multi-Channel and TurboFlow Technology from Thermo Fisher Scientific Group, Waltham, Massacheusetts, USA). In this system, two HPLC columns are operated simultaneously with staggered sample elution and column regeneration steps to maximize mass spectrometer usage and reduce lag time between elution from columns (Fig. 9.3). Other innovative approaches to improve throughput have been undertaken [74]. Hoofnagel and coworkers developed a flexible rubber gasket capable of sealing two 96-well plates together and quantitatively transfer the organic layer contents of one plate to another. A workflow involving a 96-well plate format was developed by a combination of the transfer gasket and a dry-ice acetone bath to freeze the aqueous infranatant followed by LC-MS/MS analysis of the transferred organic phase [74].

FIGURE 9.3  Multiplex Liquid Chromatography System (A) Two LC systems operating in a sequential injection sequence with no overlap between the lag times (no multiplex) and (B) two parallel LC systems staggered to achieve minimal time lag between eluting analyte of interest coupled to a single mass spectrometer. Note that by multiplexing the LC channels, mass spectrometer idle-time is reduced and throughput is improved.

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Mass detection can be achieved by atmospheric pressure chemical ionization (APCI) or electrospray ionization (ESI) methods. Derivatization by Diels-Alder reaction with substituted phenyl-1,2,4triazoline-3,5-diones (PTAD) improves sensitivity of 25(OH)D LC-MS/MS detection by a 100 fold [71,75]. PTAD addition to the 1,3-diene system common to vitamin D and its metabolites can lead to two isomeric products. Depending on the choice of the chromatographic column the different isomer may or may not separate. If no interferences are observed, choosing column that does not result in vitamin D-metabolite-PTAD isomer separation may help gain sensitivity in the method (Fig. 9.4). A differential mass-tagging approach along with multiplex HPLC was used to improve throughput by fivefold by the Mayo Clinic reference laboratory [72]. In this method (Fig. 9.5), five different barcoded patient samples are extracted and derivatized, each with a unique PTAD derivative. Then the five uniquely tagged patient samples are mixed and analyzed in a single analytical injection. Data output

FIGURE 9.4  (A) Schematic representation of the Diels-Alder derivatization reaction for 25(OH)D3; (B) Structures of PTAD derivatives used in the differential mass tagging approach. Adapted from Netzel BC, Cradic KW, Bro ET, Girtman AB, Cyr RC, Singh RJ, Grebe SK. Increasing liquid chromatography-tandem mass spectrometry throughput by mass tagging: a sample-multiplexed high-throughput assay for 25-hydroxyvitamin D2 and D3. Clin Chem 2011;57:431–440. [72]

Adapted from Netzel BC, Cradic KW, Bro ET, Girtman AB, Cyr RC, Singh RJ, Grebe SK. Increasing liquid chromatography-tandem mass spectrometry throughput by mass tagging: a sample-multiplexed high-throughput assay for 25-hydroxyvitamin D2 and D3. Clin Chem 2011;57:431–440. [72]

3 CLINICAL UTILITY AND QUANTITATION OF VITAMIN D

FIGURE 9.5  Schematic Representation of the Automated Differential Mass Tagging Approach to Improve Throughput of 25(OH)D LC-MS/MS Assay

193

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CHAPTER 9  Vitamin D metabolite quantitation by LC-MS/MS

from five PTAD-25(OH)D adducts are assigned to the respective patient samples based on original barcodes. In a high-volume clinical laboratory, differential mass-tagging combined with multiplex HPLC can provide significant gain in throughput. NIST has developed a standard reference material (SRM972) for assay standardization purposes. Additionally, deuterated and C-13 labeled internal standards and certified reference materials are commercially available (Cerrilliant, Sigma-Aldrich, St. Louis, MO, USA) and have been used in several methods [61]. However other lipophilic compounds like tetrahydrocannabinol can be successfully used as an internal standard [56]. Calibrators and QC materials can be prepared from reference materials in drug free serum or in phosphate buffered saline with or without albumin [61]. Commercial ready-touse calibrators are also available (Chromsystems Instruments and Chemicals, Gräfelfing, Germany).

3.3 C3-EPI-25(OH)D 3.3.1  Biochemistry and Clinical Utility The C3-epi-25(OH)D3 and C3-epi-25(OH)D2 in infants was first reported by Singh and coworkers in 2006. Epimerization at the C3 position of 25(OH)D gives rise to a structurally identical molecule except for differential asymmetry on carbon 3. It has been hypothesized that epimerization to C3-epi-25(OH) D is a result of an immature hepatic vitamin D metabolism common in infants. Studies evaluating calcemic effects of C3-epi-1,25(OH)2D, limited to invitro models, show that it can bind to VDR with a lower affinity compared to 1,25(OH)2D and can stimulate downstream gene transcription. Wide variation in C3-epi-25(OH)D concentration of up to 9–60% of total 25(OH)D has been observed in infants [24] and a concentration of ∼3.3 ng/mL has been reported in adolescents and adults aged 1–94 years. Failure to separate C3-epi-25(OH)D from 25(OH)D will result in an overestimation of “true” 25(OH)D concentration. An overestimation of 9% infants ( 300 g/mL. Two well documented hereditary defects in vitamin D metabolic pathway are, vitamin D-dependent rickets type I (VDDR I) and type II (VDDR II). VDDR I is caused by a reduced function of the CYP27B1 leading to muscle weakness and rickets. Rickets in VDDR I can be treated by a normal physiologic dose of 1,25(OH)2D3. VDDR II comprises a range of VDR gene mutations associated with an early onset of severe rickets and characteristic alopecia (spot baldness) [89]. Substantial doses of vitamin D analogs and calcium supplementation is usually required for the treatment; however, the response to therapy is sometimes variable. The response to 1,25(OH)2D therapy can be monitored by measuring serum and urinary minerals and PTH. Loss of CYP24A1 function results in a build-up of the active hormone, 1,25(OH)2D. Persistent hypercalcemia with elevated serum 1,25(OH)2D and suppressed or low serum PTH should prompt 25(OH)D/24,25(OH)2D ratio analysis to rule out a CYP24A1 mutation [26]. Clinical utility of quantitation of vitamin D and its metabolites are summarized in Table 9.1. 1,25(OH)2D circulates in very low concentrations (15–65 pg/mL) in normal subjects and can be challenging to quantify due to the high demand placed on sensitivity and specificity of the assay technique.

3.4.2  Assay Procedure The sample preparation procedure for a commonly used method in our and other clinical laboratories [54] is very similar to the IDS immunoassay discussed earlier. Due to the relatively low circulating concentration of 1,25(OH)2D immunoenrichment with a solid phase bound antibody greatly enhances the sensitivity and specificity of the method. Compared to 25(OH)D fourfold to fivefold larger sample volume (400–500 µL) of serum is usually used. The first step is the separation of the vitamin D metabolites from VBP. Solvents/solvent mixtures similar to that used in 25(OH)D quantitation are used (Fig. 9.2). SPE can also be used to obtain a delipidized, deproteinized sample extract. Methods may or may not use an immunoenrichment step for 1,25(OH)2D. PTAD derivatization has been used by almost all LC-MS/MS methods to enhance sensitivity. Improved sensitivity of contemporary mass spectrometers have enable successful quantitation of 1,25(OH)2D without immunopurification [62]. In one method, a combination of SPE and fixed-charge derivitization using PTAD chemistry was found comparable to immunoextraction technique. C18 or equivalent chromatographic columns (50 mm) are used and the mass spectrometer is commonly operated in the ESI mode.

3.5 24,25(OH)2D

3.5.1  Biochemistry and Clinical Utility Circulating 1,25(OH)2D constitutes only a minor fraction in picomolar concentrations of vitamin D related metabolites compared to 25(OH)D, which circulates in nanomolar concentration in nonvitamin

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D-deficient normal adults. Another dihydroxylated metabolite that is present approximately 7–35% of 25(OH)D concentration is 24,25(OH)2D. 24,25(OH)2D does not have a physiologically relevant role but is a surrogate marker for measuring CYP24A1 activity. The role of CYP24A1 enzyme in calcium homeostasis was demonstrated in CYP24A1 gene knock out mouse models. These animals developed severe hypercalcemia, hypercalciuria, and biochemical evidence of impaired vitamin D metabolism [90]. CYP24A1 biallelic mutations in humans leading to a nonfunctional CYP24A1 enzyme were first identified as the cause of idiopathic infantile hypercalcemia where an inability to metabolize the active hormone, 1,25(OH)2D results in vitamin D supplementation associated toxicity [91]. Since then several more families with mutations in the CYP24A1 gene have now been described as the cause of hypercalcemia, elevated 1,25(OH)2D, hypercalciuria, and nephrolithiasis commonly seen in these patients [26,63,92–98]. The prevalence of this recessive genetic defect is still unknown but appears to be higher than originally thought. Genotype-phenotype correlation studies suggest a variable penetrance and that haploinsufficiency is not associated with CYP24A1 deficiency [99]. A 25(OH)D/24,25(OH)2D ratio of 99 or greater identifies patients with CYP24A1 mutations [26,63,99]. In patients presenting with hypercalcemia and vitamin D toxicity, 24,25(OH)2D measurement can aide in differentiating between an iatrogenic versus genetic cause of hypercalcemia [26,69]. It is important to note that 24,25(OH)2D measurement alone provides no useful information about CYP24A1 activity because of a strong linear correlation between 25(OH)D and 24,25(OH)2D. Therefore 24,25(OH)2D must be interpreted with a concomitant 25(OH)D measurement performed on LC-MS/ MS platforms since current immunoassays cannot differentiate between 25(OH)D and 24,25(OH)2D. Even a very low 24,25(OH)2D value alone is not clinically sufficient to identify a patient with CYP24A1 mutation, since it could be merely indicative of low circulating 25(OH)D levels. As a hypothetical example in a patient with 25(OH)D = 5 ng/mL and 24,25(OH)2D = 0.5 ng/mL shows normal CYP24A1 function, whereas in a patient with 25(OH)D = 80 ng/mL and 24,25(OH)2D = 0.5 ng/mL CYP24A1 mutation is highly likely. Of note, a 25(OH)D concentration < 20 ng/mL, an increase in 25(OH)D/24,25(OH)2D is observed (Fig. 9.6) [63,99]. A role of 25(OH)D/24,25(OH)2D as a biomarker of optimal response to vitamin D supplementation has been proposed [64]. However, based on currently available scientific information 25(OH)D/24,25(OH)2D measurement is indicated only in patients presenting with persistently elevated 1,25(OH)2D, hypercalcemia along with suppressed PTH with other causes of hypercalcemia having been ruled out [26,54,63,64,99,100].

3.5.2  Assay Procedure Since 24,25(OH)2D circulates at a much higher concentration compared to 1,25(OH)2D, immunoenrichment is not required to accurately quantitate 24,25(OH)2D. Sample preparation steps are similar to 25(OH)D (Fig. 9.2). Methods with and without PTAD derivatization have been described [64,100]. Clinical laboratories measuring 25(OH)D by LC-MS/MS currently should be able to simultaneously quantitate 25(OH)D and 24,25(OH)2D by adjusting chromatographic times, optimizing ion-pairs and adding a set of calibrators and the corresponding deuterated analogs of 24,25(OH)2D2 and 24,25(OH)2D3. The calibrators can be prepared in drug free and charcoal tripped human serum or in phosphate buffered saline supplemented with 1–25 (w/vol) of albumin. It may be necessary to chromatographically separate the two isomers of 24,25(OH)2D-PTAD adduct as an interference from a closely related, 25,26(OH)2D that has the same fragmentation pattern as 24,25(OH)2D, with major isomer has been observed in our laboratory. Interference from 1,25(OH)2D-PTAD is unlikely to be a problem due to low circulating concentrations of 1,25(OH)2D and fragmentation pattern different from 24,25(OH)2DPTAD. Chromatography can be performed on 50 mm high pressure liquid chromatography (HPLC) or

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CHAPTER 9  Vitamin D metabolite quantitation by LC-MS/MS

FIGURE 9.6  (A) Association between 25(OH)D/24,25(OH)2D in normal subjects (black circles) and in patients with CYP24A1 mutations (Red triangles). A slight increase in the 25(OH)D/24,25(OH)2D is observed at 25(OH)D 99% is preferred when available. Careful evaluation of materials, used both for the calibrator and internal standard should be assessed when methods of multiple steroid hormones are used thus ensuring that impurities from the standards do not include other steroid hormones evaluated in the method. For example, it has been anecdotally noted that in estrone (E1) standards can contain trace amounts of estriol (E3). In a method evaluating E1 and E3 together, a positive bias could be introduced into the E3 results due to the presence of E1 in the E3 standard. Steroid hormones are nonpolar and hydrophobic; nevertheless, there can be an issue with crystalline powders taking on moisture during storage. The storage of crystalline materials should be carefully considered to avoid the addition of moisture. Materials should be stored in line with the certificate of analysis (COA) provided by the vendor. Consultation with vendor and evaluation of moisture content on a routine base is needed if long-term storage is necessary.

2.2  STANDARDS PREPARATION Errors in the preparation of standard solutions can result in significant bias in final patient results, and as a consequence certain parameters should be careful controlled [3]. Many considerations during preparation of standard solutions are universal and considered good laboratory practice. This includes, but is not limited to: the use of gravimetric measurements, class A glassware, calibrated pipettes, and restricted use of serial dilutions. Concerns specific to steroid hormones, such as solubility of crystalline powder materials, should be carefully considered. As steroid hormones are mostly nonpolar molecules, most are hydrophobic and poorly soluble in water. As such, the solution used to reconstitute a powder should be considered to ensure that the compound is completely dissolved prior to use; typically, an organic solvent, such as methanol or ethanol is used. After reconstitution, ample time with gentle shaking or rocking should be allowed to ensure that the solid is completely dissolved prior to the preparation of additional solutions from this solution. Incomplete incorporation of the crystalline powder into solution prior to future transfers and dilutions will result in inconsistent and inaccurate concentrations of dilutions of the stock solution.

2 Calibration and quality control requirements

207

FIGURE 10.2  Steroid Hormone Pathway

208

CHAPTER 10  Steroid hormones

Careful control of environmental conditions is needed, as temperature-dependent volume changes of solvents can occur especially with the use of volatile solvents that are used to prepare standard solutions. Controlling the temperatures of the standard and working solutions will help to ensure that proper volumes of solutions are transferred. A constant temperature, typically 20oC, consistent with the rating of the glassware and pipettes, should be maintained. This is executed through the use of a carefully monitored and maintained water bath. Additionally, due to the volatility of the solvents used in preparation of the standards, steps should be taken to minimize, and if possible avoid, evaporation of the solvents, as evaporation will alter the concentration of standard solutions and potentially introduce a measurement bias. Materials should be stored in containers with caps that create a sufficient seal to avoid evaporation and should be left uncapped for the minimal time needed to make a transfer. If materials are to be stored long-term in volatile solvents, gravimetric measurements of the containers with the solution should be considered both before and after storage as a way to monitor evaporation loss during storage and determine if concentration corrections are necessary. If standards are lightsensitive, (e.g., estradiol), amber glassware should be used and steps should be taken to avoid light exposure.

2.3 CALIBRATORS The selection of the calibrator concentration range should be tailored to the clinical application of the selected method, and depending on the application, this could be significantly different [3,4]. Careful consideration should be taken to ensure that the concentration range applicable to the clinical application is adequate and should extend beyond the lower and upper physiological range. Suggestions for the number of calibration points and spacing are the same with any other clinical method as previously discussed in earlier chapters [4]. However, with steroid hormones the concentration range typically covers several orders of magnitude; for example, estradiol can vary from < 1 pg/mL in postmenopausal women on aromatase inhibitor treatment for breast cancer versus pregnant women and women on IVF with values greater than 4000 pg/mL. Additional calibration points may be advantageous beyond the minimum recommendation to adequately cover the large dynamic range required due to the varied clinical applications of steroid hormone measurement. Additionally, a validated dilution procedure for samples beyond the established calibration curve should be considered for the occasional sample that will fall outside the established concentration range. Due to the large dynamic range, weighted regression may be needed to avoid the highest calibrator points dominating the slope of the curve and should be evaluated during method development [4]. If panels of steroids are measured in one method, the dynamic range not only within an analyte of interest but also between analytes has to be considered. For example, testosterone is measured in ng/dL and estradiol in pg/mL, and even with these significant differences in concentration, a single calibration curve with all analytes of interest in the same mixture is encouraged to minimize sampling time spent on individual calibrators. It has been recommended that preparation of calibrators should be done in matrix-matched materials to avoid introduction of a bias resulting from matrix differences among patient samples and calibrators [3,4]. Typical surrogate matrices used in steroid hormone analysis in serum include, but are not limited to, bovine serum albumin (BSA) and charcoal-stripped serum. Commercially available synthetic urine is typically used in analysis of steroid hormones in urine. Careful evaluation is needed, as problems have been noted with some surrogate matrices. Charcoal-stripped serum is prepared with the addition of activated charcoal to a serum pool, and then the charcoal is removed through filtration.

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Bias can be introduced if residuals of charcoal are present after filtration. Residual charcoal can absorb the spiked standard material, thereby resulting in an inaccurate concentration of the steroid hormones in the calibrator. Additionally, if BSA is used as a surrogate matrix it should be evaluated for residual steroid hormones. Bioavailable steroid hormones also bind to albumin, and as a result, trace amounts of steroid may be present in BSA. Careful evaluation of a surrogate matrix should be made prior to use to ensure that it will not introduce unaccounted for the concentrations of the analyte of interest or deplete the added concentration. Additionally, it should not be assumed that the surrogate matrices will remain consistent between manufactured lots. Evaluation should be made each time a new lot is used to prepare calibrators. Calibrator preparations using matrices other than unadulterated native serum should be evaluated and compared to clinical samples for differences in recovery and ion suppression [7]. Depending on the extent of sample preparation and extraction of the analyte of interest from the matrix prior to analysis by mass spectrometry, a matrix match may not be necessary but rigorous recovery and ion suppression testing is needed to confirm the absence of a matrix bias of a set of neat calibrators, for example, prepared in solvent. In addition to recovery and ion suppression experiments, matrix bias for the calibrator matrix can be assessed using a commutability evaluation strategy as detailed in CLSI EP-14 [8].

2.4  INTERNAL STANDARD Isotope-dilution should be considered for steroid hormone MS methods [4]. This approach, as previously described, can help provide the most accurate and precise results. Isotopically-labeled internal standards (IS) are commercially available for most steroid hormones (typically deuterium and/or C13 labeled forms). Standards with an incorporation of a minimum of three additional mass units are recommended to avoid overlap between the isotopic envelope of the analyte and the internal standard. A full scan evaluation of an internal standard is suggested to ensure that incomplete isotopic incorporation, or that isotopic exchange, does not overlap either directly with the analyte of interest or the isotopic envelope. Careful evaluation should be carried out in the selection of an isotopically labeled standard to ensure minimal isotopic exchange occurs. While deuterium standards may provide a more affordable approach, the standard can undergo hydrogen/deuterium exchange depending on the location of the deuterium. Isotopic exchange can also occur as a result of the environmental conditions. Evaluation of the sample preparation procedure (e.g., solvents used and pH), and mass spectrometry parameters (e.g., voltages, temperatures, and solvents) on the isotopic stability of the standard is recommended. Storage stability of internal standard materials should also be considered and evaluated, as hydrogen/deuterium exchange may occur during long-term storage of materials. In the selection of the concentration of the internal standard, the approximate mid-point of the calibration curve or at close proximity to a medical decision point has been recommended [4]. This can be somewhat extreme in situations where the dynamic range is large. The internal standard should be added to all calibrators, quality control (QC) materials, and samples prior to further sample preparation. In addition, ample time for equilibration of the internal standard is important with steroid hormones that are associated with binding proteins, such as sex-hormone binding globulin (SHBG) and albumin. If added and equilibrated appropriately, the use of the internal standard will improve the accuracy and reproducibility of the measurements by accounting for any sample loss or incomplete recovery of the analyte.

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2.5  QUALITY CONTROL MATERIALS QC preparation and use should follow criteria discussed in previous chapters [4]. Again, with the large dynamic range measurements typically seen in steroid hormone analysis, additional QCs may be needed to ensure the appropriate concentrations for the clinical application are included. For steroid hormones, bulk materials can be obtained from commercial sources and pooled. Ideally, native materials without any enhancement or depletion are preferred. However, the use of only pooled, unaltered materials may be difficult especially for the analysis of several steroid hormones at one time that require specific concentrations. As a result, spiking or dilution of the matrix may be required. Pooling, spiking, and/or diluting will alter the materials and could affect the binding protein profiles in the serum. This alteration may result in the final concentrations being significantly different than expected (especially with the analysis of free steroid hormones) and should be evaluated during preparation.

3  SAMPLE PREPARATION The first step, prior to any manipulation of the sample, is to add the IS to all samples, calibrators, and QC materials. Accuracy is critical with the addition of IS solution. Volumes greater than 50 µL, dispensed by properly calibrated pipettes are recommended. For sample preparation procedures that do not include an initial protein precipitation (PPT) step [e.g., liquid–liquid extraction (LLE), solid phase extraction (SPE), or supported liquid extraction(SLE)], care should be taken to minimize the amount of organic solvent contributed by the IS solution to avoid PPT. The precipitated protein could sequester protein-bound steroids away from subsequent extraction steps, resulting in falsely low results. Additionally, ample time should be allowed for the equilibration of the IS solution with any protein-bound steroids. Testosterone has been reported to reach equilibration with the internal standard in 30 min [9]. With proper addition and equilibration time, a stable IS signal should be observed from the MS. If large variations in signal response are observed, incomplete equilibration or inaccurate addition of the internal standard solution could be the cause. Steroid hormones are bound to albumin with low affinity and to SHBG with high affinity in serum. Only a small percentage of steroids are not protein-bound, and these represent the free fraction. Due to the lower affinity of binding to albumin, these steroids are also available in vivo. Therefore, the free fraction and the albumin-bound fractions make up the bioavailable steroid hormones. Steroid hormones also form conjugates with sulfates and glucuronides, especially in urine, and are found at higher concentrations in some cases than the free steroid. Free steroid hormones can be directly measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS) but require additional procedures to isolate the free steroids while taking care not to effect the equilibrium of free and bound steroids [10]. Procedures, such as equilibrium dialysis and ultracentrifugation have been reported in literature [11–13]. Assays for free steroids have to be more sensitive due to the low concentrations that are observed in free steroid measurements. To measure total steroid hormone concentrations, the analyte of interest must be released from the binding protein or conjugate. Protein dissociation can be achieved with protein denaturing. The denaturation should break the bond between the steroid and the protein(s) without altering the analyte of interest. For testosterone it has been reported that this can be achieved at a pH of 5.5 for 30 min [9]. A hydrolysis step is needed to deconjugate the glucuronide group from the steroid hormones if total hormone concentration is required. In most cases, this is achieved with β-glucuronidase [14].

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Isolation of the analyte of interest from the matrix is recommended to reduce the complexity of the sample that is to be analyzed. Proteins, peptides, small molecules, salts, phospholipids, and other compounds can suppress or interfere with the measurements of steroid hormones by LC/MS, resulting in insufficient resolution, loss of sensitivity, reproducibility, and robustness due to ion suppression [15]. Extraction can be performed offline or online to allow for higher sample throughput. Automation steps can be considered as previously discussed. Common sample extraction procedures used in steroid hormone analysis include; PPT, LLE, SPE, and SLE. Examples of publications with different sample preparation techniques are referenced in Table 10.1. For steroid hormone analysis, PPT is most often performed by adding a high concentration of organic solvent or organic solvent plus a salt solution to the sample to separate the steroids from binding proteins and other matrix proteins. PPT is inexpensive and easily automated, however ion suppression and instrument contamination due to insufficient matrix removal may prevent the ability to achieve sufficient steroid assay sensitivity. SPE is a rapid preparation technique that employs partitioning between a mobile phase and a solid phase, similar to conventional liquid chromatography (LC). SPE cartridges containing reversed-phase sorbent are commonly used for steroid analysis. The relatively large particle size of the SPE sorbent allows for fast sample preparation. SPE is easily automated and can also be performed online, where samples interact with a SPE column immediately prior the analytical column. LLE utilizes partitioning between two immiscible liquids (e.g., aqueous and organic solvent) and is a common approach for steroid analysis, as LLE exhibits good extraction efficiency for nonpolar analytes and is more selective than PPT. Modification of the pH in LLE can be used to change the selectivity of the extraction and improve recovery. In SLE, a solid support material with a hydrophilic surface is used to retain droplets of the aqueous serum sample containing the steroids to be measured. The supported aqueous phase is then exposed to an organic solvent for extraction of the steroids, similar to standard LLE. SLE reduces the number of steps required for the preparation compared to LLE and SPE, and allows faster sample processing [16]. However, one drawback of SLE is reduced mixing and surface area for extraction as compared to LLE. The choice of sample preparation technique depends on required assay sensitivity, selectivity, and labor restraints. Many sample preparation techniques include use of multiple combinations of sample preparation approaches (Table 10.1), and most are amenable to some level of automation, such as automated SPE/SLE or use of an automated liquid handler. Many steroids are difficult to resolve chromatographically and they exhibit poor ionization in the mass spectrometer. Chemical derivatization during the sample preparation process may be used to improve chromatographic separation, increase robustness in MS fragmentation, and achieve greater sensitivity by improving ionization efficiency. Common derivatizing agents used in steroid hormone analysis include dansyl chloride, pentafluorobenzyl, picolinoyl, pyridyl, and piperazinyl. Through the addition of the derivative, ionization and chromatographic separation may improve and the molecular weight of the analyte is increased, which may result in more robust precursor ions and availability of additional mass transitions. Although derivatization can improve assay sensitivity, use of derivatization procedures can induce the formation of isomers and nonspecific product ions. Therefore, the specificity of the method will increase if the fragment ion selected for quantification is not the loss of the derivative (i.e., just the mass of the analyte you are measuring). The sensitivity and robustness of a method may be improved with the removal of derivatization reagents via an additional cleanup step. Routine clinical laboratories may choose to avoid methods that employ derivatization procedures because derivatization often increases assay labor requirements and reagent expense.

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Table 10.1  Examples of Published Transitions for Selected Reaction Monitoring of Nonderivatized Steroid Hormones by Liquid Chromatography Tandem Mass Spectrometry Steroid Hormone

Ion

Mass Transition

Number Ionization of LoQ Source Steroids (ng/L)

Analytical Sample Column Preparation

References

Androstenedione

[M + H]+

287/97 287/109; 97 287/97 287/97 287/97; 109

ESI APCI ESI ESI APCI

10 3 3 2 9

404 100 30 72 20

C18 C18 C18 C18 C18

PP PP + SPEOL SPE LLE PP + SPE

[18] [19] [20] [21] [22]

DHEAa

[M−H2O + H]+

271/197; 213

APCI

9

20

C18

PP + SPE

[23]

DHEAS

[M − H]− 367/97

ESI

1

3.6e5

C18

PP

[23]

Testosterone

[M + H]+

289/97; 109 289/97 289/97; 109 289/109 287/109; 97 287/109; 97 289/97 289/97 289/97 289/109 289/109; 97 289/97; 109 289/97 289/97; 109

ESI ESI APCI ESI APCI APCI ESI ESI ESI ESI APCI ESI APCI ESI

2 1 1 10 3 1 1 1 3 2 9 1 8 1

20 50 100 600 100 3 61 87 30 72 20 10 20 20

C18 C18 C18 C18 C18 C12 C18 C18 C18 C18 C18 C18 C18 C18

LLE LLE PP + SPEOL PP PP + SPEOL SPEOL PP + LLE PP SPE LLE PP + SPE LLE LLE LLE

[24], [25] [26] [27] [18] [19] [28] [29] [30] [20] [21] [22] [31] [32] [33]

DHT

[M + H]+

291/255 291/255 291/255

ESI ESI ESI

2 10 3

20 854 30

C18 C18 C18

LLE PP SPE

[24], [25] [18] [20]

Estradiol

[M − H]− 271/145; 183

ESI

4

2b

C8

PP

[34]

Estrone

[M − H]− 269/145; 143

ESI

4

1b

C8

PP

[34]

Estronesulfate

[M-SO3−]− 349/269 349/269

ESI ESI

1 1

8 80

C18 C18

PP SPE

[35] [36]

NOTE: The m/z of the tenth decimal position may vary slightly, based on instrument tuning. Therefore, values for m/z are given to the nearest whole number. Abbreviations: APCI, atmospheric pressure chemical ionization; CV, coefficient of variation; DHEA, dehydroepiandrosterone; DHEAS, dehydroepiandrosterone sulfate; DHT, dihydrotestosterone; ESI, electrospray ionization; LLE, liquid–liquid extraction; LoD, limit of detection; LoQ, limit of quantitation; PP, protein precipitation; SPE, solid phase extraction; SPEOL, solid phase extraction online. a Water-loss ion. b Value stated as “LOD” with CV 1000 cells per second. Mass cytometry can also be used to generate fluorescent cell barcodes for cellular phenotypes [50]. Forward and side scatter are not available in first generation mass cytometer. The second generation mass cytometer CyTOF2 can resolve more than 120 metal probes, more than 36 proteins simultaneously in a single tube. The most current generation mass cytometer, the Helios (CyTOF3) has 135 detection channels, which in conjunction with the resolving power of the TOF MS, enables monitoring of more than 40 markers per cell. CyTOF has applications in immunology, hematology, and oncology research [51,52]. Studies have focused on immunophenotyping, intracellular cytokines profiling, and characterizing phosphorylation signaling pathways [53]. Mass-tag cell bar-coding has been used on induced pluripotent stem cell reprogramming [54] and in multiple myeloma [55,56]. The major limitations of this platform include reliance on antibody specificity, sample type and preparation—purifying isotopes and attaching reporter isotopes to antibodies (which is a commercially available service through Fluidigm), vigilance in avoiding heavy metal contamination by avoiding chelators, clean time, oxidation of reporter tags, cost, and expertise required for manual analysis.

7 PCR-MS PCR-MALDI-TOF has been used to detect genetic polymorphisms in the transmission of a rapidly mutating pathogen, hepatitis C virus [57–59]. PCR-qTOF MS has been applied to the identification of microbes and the quantitation of virus [60]. Abbott Technologies’ IRIDICA platform, the second generation of Ibis Biosciences’ Plex-ID, relies on MS detection of amplicons directly from whole blood, bronchial lavage, and other bodily fluids for microbial identification. This technology targets critically ill patients by reducing the time of bacterial, fungal, and viral identification from days and weeks to hours and including select antibiotic resistance markers. There are different workflows for the types of identification; bacterial workflow differs from the fungal and the viral. The throughput is low, six samples total per 8 h, and a control sample should be

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included with each batch. Also, there are some challenges with complete identification in mixed bacterial infections. A similar approach is used to rapidly identify microbes from blood cultures [61]. And a type-specific quantitative detection of human papillomavirus (HPV) by a PCR-MS based method was developed and evaluated for cervical cancer screening [62]. PCR-MS based approaches are not trivial to implement. PCR requires appropriate primers for amplification of oligonucleotides of unique masses, DNA extraction, vigilance in technique to reduce and avoid DNA contamination, optimization, and thorough evaluation and analysis to link the oligonucleotides to the pathology of interest. However, once these parameters are set up, PCR-MS can provide reliable and accurate results.

8  FRONT END—SAMPLE PREPARATION AND COLLECTION/AMBIENT IONIZATION/REAL TIME ANALYSIS The evolution of clinical mass spectrometry is a balance between utilizing technologies that exist and creating technologies that fit clinical needs. A large portion of the turnaround time from collection to result is sample preparation and/or cleans up with minutes-long LC methods. Hence, with easier, faster, or standardized sample collection and preparation with removal of LC, the time to result would be reduced. This can be accomplished through efforts in non- or less-invasive sampling techniques, ambient ionization approaches, and real-time analysis [63].

8.1  SAMPLE PREPARATION/SAMPLE COLLECTION The push to gather more information from small sample volumes has spawned creative exploration of different sample types, collection methods or devices for sample collection, and methods of preparation. In addition to previously mentioned immunocapture and SISCAPA strategies to pull out analytes of interest from small sample volumes, there are specialized sample collection devices. A few examples include blood spot collection devices that wick the plasma away from the red blood cells, devices that can collect a fixed, small volume of sample on the order of 10–20 µL, and breathe collection devices that have a filtered mouthpiece to capture aerosols. Non- or less-invasive sample types, such as breath, saliva, tears, or surface swabs or direct solvent extraction may play a larger role in the near future.

8.2  VOLATILE SAMPLING AND REAL TIME ANALYSIS Analysis of breath samples is used for drug detection and has been used in forensic applications with flowing-afterglow (FA) and selected ion flow tube (SIFT) MS for real-time breath printing and metabolomics [64,65]. Breath analysis has implications in the clinical lab [66,67]. Chemical signatures of volatile organic compounds (VOCs) on breath from lung infections, acetone (a reflection of blood sugar levels), acetonitrile (smoking), and ethanol have been evaluated [68]. Real time breath analysis can be used to monitor the health status of CF patients through exhaled volatile biomarkers of Pseudomonas aeruginosa infection [69] or indicators of acidity of the airway mucosa [70]. These breath studies have been conducted in real time with SIFT MS and proton transfer reaction (PTR) MS [71,72]. These MS approaches are faster than GC and also allow collection of breath into bags or onto traps/ filters for later analysis. SIFT-MS and PTR-MS are both chemical ionization (CI) methods that yield

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little to no fragmentation. And quantitation is accomplished via internal ion ratios, similar to analysis of MRM data from triple quad MS platforms. Similar to breath testing, a group developed a man-portable membrane inlet mass spectrometer (MIMS) to generate “odorprints” by detecting odors generated from humans, breath and sweat, in a confined space [73]. However, generation of “odorprints” is not time efficient due to the large sampling space of a room. MIMS was also evaluated for narcotics, explosives, and chemical warfare agents on different membranes for use in homeland security measures. MIMS can be used for water, air, and some solvents for real-time analysis of VOCs and some small molecules [74].

9  AMBIENT AND DIRECT IONIZATION APPROACHES 9.1 DESI Desorption electrospray ionization (DESI), developed in Graham Cook’s laboratory and commercially available by Prosolia, Inc., relies on solvent extraction directly on the sample surface for localized information. DESI, which has been shown to ionize large proteins and complexes [75], is primary used on tissues in imaging mass spectrometry. Iterations of DESI hold more promise in clinical applications. DESI combined with solid-phase microextraction was used to screen and quantitate drugs in urine [76]. Single droplet microextraction with DESI MS has been shown for trace analysis of methamphetamine in aqueous solution [77] and can be applied to other small molecules. Transmission mode (TM) DESI is more conducive to throughput, by reducing optimization of solvent delivery and sample introduction angles through the use of a mesh material as a sample substrate, and placing the mesh with 1 µL of sample in-line with the ESI source and mass spectrometer inlet [78,79]. However, TM-DESI has mainly been used for proof of principle studies of standards in solution, not for biofluid samples with complex biometrics.

9.2  PAPER SPRAY LC-MS/MS analysis of dried blood spots from heel sticks have been used for biochemical genetics since the 90s [80,81]. Punch disks of the dried blood spots, used in an attempt to normalize the amount of sample, are extracted in 96 well plates then introduced into a triple quad mass spectrometer. The next step to minimize sample handling would be to combine the dried blood spots with paper spray. Paper spray (PS) MS by Prosolia, Inc. is a combination of paper chromatography and electrospray ionization, and has been used to analyze immunosuppressant’s in whole blood collected on triangular filter paper [82–84]. In PS MS the dried sample spot undergoes slight separation based on the wicking of the solvent through the filter paper. A voltage is applied to the filter paper and the pointed tip of the triangle, closest to the mass spectrometer inlet, is the location where sample enters the mass spectrometer. PS MS has also been used to detect microorganisms [85], tobacco derived nicotine alkaloids [86], and other molecules from biofluids, blood, urine, and saliva in both biofluid and dried spots [87–90].

9.3  TOUCH SPRAY Another iteration of placing analyte in the stream of the electrospray path is touch spray (TS) [85,91]. Touch spray takes advantage of existing swab sampling for collection of surface molecules. Unlike the pointed filter paper in paper spray, the swab is physically placed in the spray path, partially obstructing

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the spray, analytes caught in the stream get ionized, and travel into the mass spectrometer for detection. This has been used to capture the phospholipid and glycerophospholipid profile of Streptococcus pyogenes responsible for strep throat infections. However, in order to be conductive, the swab used in the studies has a metal handle, which would constitute a significant change in cost and clinical practice. Similar to DESI, Zhang et al. developed internal extractive electrospray ionization (iEESI) [92], in which charged solvent extraction through a sample (1–100 mm3) travels to the mass spectrometer. The authors claim sampling occurs deeper than merely the surface. Direct analysis in real time (DART) MS, developed in 2005, is an ambient ionization approach that does not require sample preparation. Ionization occurs directly on the sample, which can be liquid or solid as in pills or hair. DART MS has been used to detect or quantify drugs in toxicology and forensics, steroid hormones in clinical trials, fragrances, food contaminants, and pesticides with masses up to 800 Da [93]. A study used DART to analyze changes in the mouse skin metabolome when exposed to UV B [94] and one can deduce a parallel study in humans. Laser ablation electrospray ionization (LAESI) (Protea Biosciences) uses a laser to generate a plume that gets caught in the path of electrospray and can detect masses up to 66 kDa. LAESI, another chromatography free technique, is most useful with samples that contain water and has been primarily used in toxicology. LAESI has also been used to detect drugs from human hair [95]. DART, DESI, TS, and PS can be paired with the mass analyzer that provides the mass resolution and sensitivity required for the study at hand. For example, there is DESI-LIT and DESI-TOF. The ideal dynamic range to cover biomolecules, from small molecules (10s Da) and metabolites to lipids (100s Da) to proteins (100,000s Da) needs to span ∼5 orders of magnitude.

10  IMAGING MS Imaging mass spectrometry (IMS, not to be confused with ion mobility mass spectrometry) or mass spectrometry imaging (MSI) produces a chemical snapshot, revealing the spatial distribution of molecules of one point in time. IMS is label-free and does not require the numerous steps in histological staining for tissue analysis. However, sample preparation of tissues requires cryosectioning, which requires practiced hands. In theory, any mass analyzer is capable of IMS as long as the front end is conducive to scanning over an area and the data can be processed and displayed in a two-dimensional manner.

10.1  MALDI-TOF IMAGING Since its introduction in 1997 [96], MALDI-TOF IMS has been applied to direct lipid profiling in clear cell renal cell carcinoma [97], human colorectal cancers [98], gastric and breast cancer tissues, and tissue microarrays for different cancers [99]. MALDI-TOF mass spectrometers are powerful and can image at resolution approaching single cell resolution [100]. In addition to MALDI-TOF, desorption electrospray ionization (DESI)-qTOF, DESI-IT, and TOF-SIMS [101] are IMS platforms used in research.

11  DESI IMAGING DESI, like MALDI-TOF, has been used for label-free, stain-free tissue imaging, with the advantage of ambient ionization. Detection of molecules via DESI depends on the extent of solvent permeation, sample extraction. Spatial resolution for the image is dictated by the size of the solvent droplet or the

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splash zone and ionization is affected by the spray angle. The types of molecules detected are dependent on the solvents used. DESI of biopsy tissue sections allows a deeper analysis, complementing traditional H&E staining for histopathology [102,103] distinguish two meningioma subtypes based on lipid profiles (negative mode MS), correlates well with histopathological analyses [104,105]. Most DESI studies have been conducted in negative ion mode, favoring the ionization and detection of lipids to profile tumor heterogeneity [106–109]. The turnaround time of DESI imaging is faster than histology, but slower than the iKnife. The advantages of imaging mass spectrometry include the ability to profile in situ cell heterogeneity label-free, to differentiate between healthy tissue and cancer. One major challenge with imaging mass spectrometry platforms still is translating the chemical or molecular information to biological information. Parallel processing with H&E and other immunohistochemical staining and close collaboration with histologists/pathologists are required at least initially, to corroborate the imaging data generated from mass spectrometry. Co-registering the immunohistochemical (IHC) images helps connect the two. Tissue samples for imaging mass spectrometry require either surgery or biopsy, which is invasive, and the majority of IMS results are retrospective. The turnaround time is days to weeks, and data analysis requires a database or library of spectra. Data files are large, so computing power, data management, and programs that support large files are concerns. Complementary to histology, laser micro dissection and mass spectrometry was used to evaluate a renal biopsy for monoclonal IgG deposition disease [110]. And less invasive means for cellular collection, needle biopsy or fine needle aspirate smears can be used with DESI-MS imaging for complementary molecular information toward diagnosis [111,112]. One technique that demonstrates direct clinical application and is already being used in operating rooms is rapid evaporative ionization mass spectrometry (REIMS) [113]. The intelligent knife (iKnife) takes advantage of the laser used in surgery, was developed by Zoltan Takats’s group, and is commercially available through Waters Corporation. The iKnife provides real time feedback to surgeons, enabling on-the-fly decisions about margin control. The boundary between tumor and healthy tissues is detected via comparison of real time laser ablation mass spectra during surgery to a database of spectra profiles of healthy and diseased tissues [114,115]. Surgeons with the iKnife rely on mass spectra to distinguish the boundaries of tumor versus healthy tissues and/or different cancer grades. REIMS also has utility in microbial identification [116].

12  ION MOBILITY MASS SPECTROMETRY Ion mobility mass spectrometry, separation of molecules in the gas phase, has applications in breath analysis [117], “skin-sniffing” or surface analysis, and lipoprotein particle size and concentration analysis [118].

13 PROTEOMICS Targeted proteomics has made its debut in the clinical lab and can be accomplished by immunoaffinity capture and triple quad LC-MS or MALDI-TOF MS as a combination of SID and MRM. Targeted proteomics requires rigorous front end analysis and a level of expertise to ensure that the monitored peptide/s reflect the amount of protein present. The immunoaffinity capture requires the generation of specific antibodies to the biomarker, or analyte of interest. This approach is dubbed mass spectrometric

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immunoassay (MSIA) [119–121]. Initially a biomarker/protein that has clinical significance or differential expression—quantitative results will guide therapy, treatment, or clinical decision making—is selected. This protein or peptide must contain a unique peptide/be unique, with little to no modification sites for accurate quantification. Second, detectability (coverage) of the candidate biomarker must be evaluated via mass spectrometry. Third, candidate peptide/s and fragment peptides are selected to serve as a proxy for the entire protein. And a synthetic labeled peptide must be synthesized to serve as an internal standard for quantitation. Fourth, the protocol and selection of peptide/biomarker must be evaluated in context of patient samples and clinical utility. With this approach, protein isoforms/variants can also be monitored [122–126]. With the increasing use of biologics, stem cell therapy, protein based therapies; this approach will become more and more prominent in clinical mass spectrometry. The main advantages of this approach are avoiding the interference from endogenous immunoglobulins in immunoasssays, and sensitivity, or being able to quantitate down to picomolar quantities. The main challenge or bottleneck lies in selecting a peptide to serve as a protein surrogate, or biomarker discovery and validation. Another possible disadvantage is the immunoaffinity enrichment step in the development process if the protein has different forms or if protein degradation products containing the epitope are present [127]. Targeted quantitative proteomics may directly affect patient care in the near future, as a predictor of patient response to therapy. Assay development for clinical proteomics is not trivial, in addition to the limitations/challenges for mass spectrometry. Additional exciting advances in mass spectrometry may have future clinical impact. Electrochemical microfluidic chips to detect short-lived drug metabolites [128], slug-flow microextraction [129] deal with generating MS data from small volumes of sample. Liquid electrospray laser desorption/ ionization (liquid EDLI) allows native protein ions directly from aqueous solutions and biofluids (ref). However, most of the emerging technologies in clinical mass spectrometry are qualitative. The demand for quantitation has been addressed by Liu et al. by coating the inside of glass capillaries with internal standard prior to use for trace analysis in complex biological mixtures [130]. And the field is quickly moving toward quantitation [131].

14  FUTURE IMPLICATIONS There is a demand in clinical mass spectrometry toward quantitation of multiple analytes from small volumes of sample, drop of blood or tear drop, swabbing or skin sensors, or noninvasive breath collection. Monitoring multiple analytes falls in the realm of omics studies, and with metadata can be put in the category of personalized medicine. The push for untargeted omics approaches, such as the Human Longevity, Inc. (HLI) of JCVI, yields massive amounts of data, shifting the bottleneck to data processing, and management. However, with the complexity of gene expression profiles, temporal changes, splice variants, posttranslational modifications of proteins, and the interplay of metabolites with the microbiome, monitoring all genes, proteins and metabolites over time is not a trivial task. Despite the ability to acquire data, we yet don’t know the full meaning of all this information. The search for biomarkers elevated in disease continues. Advantages of the evolving platforms in clinical mass spectrometry include decreased cost of healthcare overall, the capacity for easy, high throughput assays, accurate results, and improved turnaround time.

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Technologically, these future implications push for an increase in detection sensitivity and reliable and reproducible ionization from any biological matrix. Ambient sampling/ionization reduces the reliance on liquid chromatography for separation. This will generate a lot of data, which also comes with the responsibility of educating the public appropriately.

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Index A Absolute mass spectrometry detector signal, 21 Accuracy, 1, 9, 65, 72, 90, 102, 154, 173, 217, 249 Acetaminophen, 109, 152 Acetonitrile, 42, 55, 168, 186, 189 Aldosterone, 219 Aliquot, 9, 11, 21, 89, 104, 139 Ambient ionization techniques, 18 Amitriptyline, 153, 169 EI spectra of, 153 AMR. See Analytical measuring range (AMR) Anaerobic bacteria, 237 Analysis, 147 creatinine, 8 direct probe, 139 ESI-TOF, 253 GC-MS, 214 applications for steroid, 1 immunosuppressant compounds, 88 larger biomolecules, 251 MALDI-TOF MS, 238, 240, 241, 253 methylmalonic acid, 78 nucleotide, 255 opioid, 119 proteins and peptides, 19 qualitative screening, 147 quality assurance for specimen, 103 sensitivity of, 25 steroid hormone, 208, 210 synthetic cannabinoids, 251 testosterone, 221 toxicological, 109 urinary free cortisol, 100 using APCI, 2 volatiles, 139, 213 Analyte extraction, 23, 142 Analytes, 1, 2, 6, 20, 23, 40, 45, 54, 56, 101, 132, 145, 184, 205, 213, 247, 262 Analytical instrumentation, 19 Analytical limitation, 24 Analytical measurement range (AMR), 66, 171 Analytical performance characteristics, of different mass analyzers, 32 Analytical sensitivity, 65 Analytical specificity, 66 Analyzer, types of, 134 hybrid, 138 ion trap, 134–135  

orbitrap, 138 quadrupole mass filter, 135 single quadrupole analyzers, 135–136 triple quadrupole analyzers, 136–137 time of flight, 137 Androgens, measurement in adult, 218 Antibiotic resistance, 263 Antibiotic therapy, 235 Antimicrobial susceptibility testing (AST), 241 Antirachitic compound, 181 Antiretroviral (ARV) drugs, 252 APCI. See Atmospheric pressure chemical ionization (APCI) APPI. See Atmospheric pressure photoionization (APPI) ARV drugs. See Antiretroviral (ARV) drugs Assay characterization experiments, 22. See also various techniques Assay design/test life cycle considerations, 170 business management, 170 patents, 170 regulatory assessment, 170 Assay validation, 152. See also Validation goal of, 152 parameters of, 152 accuracy, 154 analyte carryover, 155 analyte recovery, 155 analyte stability, 155 linear response, 154 lower level of quantitation, 152 method comparison, 154 precision, 153 sensitivity, 152 specificity, 152 recommendations and guidelines, 152 AST. See Antimicrobial susceptibility testing (AST) Atmospheric ionization techniques, 22 Atmospheric pressure chemical ionization (APCI), 2, 22, 192, 247 Atmospheric pressure ionization (API) techniques, 27 Atmospheric pressure photoionization (APPI), 3, 22, 247 Automatable assays, 17 Automated analyzer, 17 Automated differential mass tagging approach 25(OH)D LC-MS/MS assay, throughput improvement of, 193 Automated immunoassay analyzers, 17 Autosampler maintenance, 102 Average ion suppression, 175

277

278

Index

B Bacillus anthracis, 237 Bacteriology, performance of MALDI-TOF MS systems, 235–237 Batch-based analysis of samples, 90 Benzodiazepines, 56, 145 chromatography of, 147 Benzoyecgonine calibration curve, 154 linearity of response, 154 Biochemistry assays, 17 Biological matrix, 18, 21 Bio-Rad REMEDi drug profiling system, 125 Biosafety level 3 (BSL-3), 238 Blackbox, 17 Bland-Altman plot, 86, 173 Boiling point, 23 Bone mineralization effects of vitamin D, physiological role, 195 Bovine serum albumin (BSA), 208 as surrogate matrix, 209 Broad spectrum drug screening, 123, 124 biphenyl, 123 matrix effects, 126 method development, 123, 126 Bio-Rad REMEDi drug profiling system, 125 GC-MS full scan method, 125 rational for, 125 method validation, 126 MS scan modes used for, 114 pentafluorophenyl, 123 postimplementation monitoring, 127 primary stationary phase used for, 123 TOF mass spectrometers, 125 Bruker Biotyper library, 237 Bruker system, 236 BSL-3. See Biosafety level 3 (BSL-3) Burkholderia pseudomallei, 234, 237 Busulfan, 167 method comparison by LC-MS/MS to GC-MS, 174

C Caffeine, 152, 224 Calcitroic acid, 195 Calcium binding protein, 195 Calibration, 8, 21, 67, 83, 87, 95, 141, 155, 208, 249 insufficient, 249 materials, 83 Calibrators, 67, 85–87, 208 concentration range, for clinical application, 208 preparation of, 79, 208 selection of, 208

Campylobacter jejuni, 241 Cannabinoids chromatography and detection, 147 derivatized in order to improve, 147 methylation, 147 silylation, 147 CAP. See College of American Pathologists (CAP) Cathinones, 251 Center for Medicare and Medicare Services (CMS), 71 Centrifugation, 39, 144 C3-EPI-25(OH)D assay procedure, 194 biochemistry and clinical utility, 194 Chaperone protein, 188 Chemical ionization (CI), 265 technique, 26 Cholecalciferol, 181 Chromatographic elution, 22 Chromatography, 24, 109, 140, 171, 251. See also specific techniques carrier gas flow rate, 141 column and temperature gradient, 140 filament start delay, 140 solvent delay, 140 split injection mode, 140 splitless injection mode, 140 Chronic kidney disease (CKD), 196 CI. See Chemical ionization (CI) CKD. See Chronic kidney disease (CKD) CLIA. See Clinical Laboratory Improvement Act (CLIA) Clinical and Laboratory Standards Institute (CLSI), 172 documents, 64 linearity of quantification procedure, 88 Clinical Laboratory Improvement Act (CLIA), 172 Clinical Laboratory Improvements Amendments, 64, 67, 72 verification of performance elements, 64 Clinical mass spectrometry, 1 analytical power of, 1 analyzers used in, 3 hybrid tandem mass analyzers, 5 ion trap mass analyzers, 5 quadrupole mass analyzers, 4 time-of-flight mass analyzers, 4 basic concepts, 1 challenges ionization techniques used in, 3 ion sources for, 2 atmospheric pressure chemical ionization (APCI), 2 atmospheric pressure photoionization (APPI), 3 electrospray ionization (ESI), 2 matrices used in, 9 potential pitfalls and challenges, 8 external quality assessment and quality control, 8–9

Index

matrix effects and interferences, 9–11 practical considerations, 12–14 technical hurdles, 11–12 resolving power, definition, 1 schematic diagram of, 2 segments of, 2 Clinical toxicology, 251 Clomipramine broad spectrum drug screening, 127 identification of, 127 Clostridium difficile, 241 CLSI. See Clinical and Laboratory Standards Institute (CLSI) Coagulase-negative staphylococci (CoNS), 237 Coefficient of variation (CV), 65 College of American Pathologists (CAP), 176 accreditation standards, 64 inspection checklists, 68 elements related to mass spectrometry assay validation, 68–69 urine toxicology survey, 127 Collision associated dissociation (CAD), 30 Collision chamber, 30 Collision-induced dissociation (CID), 4, 30 Column dimensions, 22 Community-acquired pneumonia, 236 Competency assessment, 71 requirements, 71 Compromised efficiency, 22 CoNS. See Coagulase-negative staphylococci (CoNS) Controls, preparation of, 79 Cost per test (CPT), 168 Cost vs. performance of the mass analyzers of interest to the clinical laboratories, 32 CPT. See Cost per test (CPT) α-Cyano-4-hydroxycinnamic acid, 232 Cyclobenzaprine, 153 EI spectra of, 153 Cyclopentanophenophenanthrene ring system, 205 illustration of, 206 CYP24A1 gene knock out mouse models, 196 CYP24A1 mutation, 197 association between 25(OH)D/24, 25(OH)2D, 198 Cytochrome P450 (CYP) 2R1, 181

D Dansyl chloride, 211 as derivatizing agents used, in steroid hormone, 211 DART. See Direct analysis in real time (DART) Database-driven toxicology, 252 Data dependent acquisition (DDA) criteria, 123 Data dependent acquisition strategies (DDA), 252

279

Data independent acquisition (DIA), 252 Data integration software packages, 91 Data review, 90–100 acceptable recovery of the internal standard, 94 calibration curves, 94 standard practice for, 95 charging, 94 chromatogram, reviewed to ensure appropriate integration of peaks, 95 common sources of reduced internal standard recovery, 93 confirmation, 93 initiate with a check of batch acceptance, 91 ionization suppression, 94 IS drift associated with, 94 process of, 91 QC values assessed following completion of calibration curve generation, 95 retention time reproducibility and alignment of retention times of analyte(s) and, 95 sample preparation-induced drift, 93 transition ratios performed by generating peak areas of, 98 use of transition ratios, 98, 100 DBS. See Dried bloodspots (DBS) DDA. See Data dependent acquisition strategies (DDA) 7-Dehydrocholesterol, 181 Dehydroepiandrosterone (DHEA), 218 Dehydroepiandrosterone sulfate (DHEAS), 218 DEQAS. See Vitamin D, external quality assessment scheme (DEQAS) Derivatization, 24 DESI. See Desorption electrospray ionization (DESI) Desorption electrospray ionization (DESI), 266 Detector, 18, 21, 111, 134, 156, 232 DIA. See Data independent acquisition (DIA) Diagnostic fragment(s), 30 Dialysis, 23 Diazepam, thermal decomposition of, 147 Diels-Alder reaction, 192 Dilution sample preparation protocol (DIL), 39 optimizing, 56 protocol, 39 Direct analysis in real time (DART), 267 Direct on-plate extraction, 240 Direct probe analysis, 139 Dispersive liquid-liquid microextraction (DLLME), 144 Dried bloodspots (DBS), 69 current draft versions of the FDA guidelines, 69 nuances, 69 as a sample type, considerations regarding use of, 69 volume per area with hematocrit, 69 Drug-free urine specimen chromatograms by SPE method, 143 Drug screening, 148

280

Index

E Efavirenz pharmacokinetics, 255 Electron impact (EI) ionization technique, 26, 131, 153 Electron transfer dissociation (ETD), 263 Electrospray ionization (ESI), 2, 21, 40, 192, 247 ESI mass spectra, 28 Electrostatic lenses, 21 Endocrinology, 24 Epimers separation, 194 Ergocalciferol, 181 ESI. See Electrospray ionization (ESI) Estriol, 249 mass resolution, 250 Estrogens, 219 ETD. See Electron transfer dissociation (ETD) Ethyl acetate, 58 European Medicines Agency’s guideline on bioanalytical method validation, 72 Europe Institute for Reference Materials and Measurements (IRMM), 205 Evaluating method performance, 48 Evaluating practicality, 48 Evaluating robustness, 49 Exact mass, 248 External quality assessment for LC-MS/MS methods, 8–9 and quality control, 8–9 Extraction, 18 efficiency, 21 Ezogabine, 169

F FA. See Flowing-afterglow (FA) Facklamia hominis, 235 Fatty acids, 24 Fentanyl, validation data for, 121, 122 18F-FDG. See 18Fluorodeoxyglucose (18F-FDG) Filamentous Fungi library, 240 Filtrate, 39 Flowing-afterglow (FA), 265 18Fluorodeoxyglucose (18F-FDG), 256 Forensic toxicology, 251 Fortification, 86 Fourier transform ion cyclotron resonance (FTICR), 31 Francisella tularensis, 234, 237 Full width at half maximum (FWHM), 249 FWHM. See Full width at half maximum (FWHM)

G Gas chromatography (GC), 111, 261 column, 24 Gas chromatography (GC)-MS, in clinical toxicology, 131

clinical applications of, 132 drug confirmation, 148 drug screening, 148 full scan method, 125 instrument selection, 133 limitations of, 132 personnel, 160 background and experience, 160 training, 160 background and experience, 160 workflows, 29 Gas liquid chromatography, 237 GC. See Gas chromatography (GC) Gram-negative bacteria, 231 Gram-positive bacteria, 231

H Headspace injection, 139 Healthcare reimbursement, 168 Hematopoietic stem cell transplant, 167 Hemolysis, 21, 175 High performance liquid chromatography (HPLC), 166 separation, quality of, 22 High resolution (HR) mass analyzers, 29, 31 High resolution mass spectrometry (HRMS), 31, 247 fundamentals, 247 future clinical applications, 255 future clinical developments, 255 hybrid instruments, 251 HPLC. See High performance liquid chromatography (HPLC) HPV. See Human papillomavirus (HPV) HRMS. See High resolution mass spectrometry (HRMS) Human papillomavirus (HPV), 264 Hybrid instruments, 5, 32, 263 CYTOF (ICP-MS), 264 Quadrupole-Orbitrap MS, 263 Hybrid tandem mass analyzers, 5 Hydrocodone, validation data for, 121, 122 Hydromorphone, validation data for, 121, 122 Hydrophobicity, 39 25-Hydroxyvitamin D assay procedure, 189–194 biochemistry and clinical utility, 189 25-Hydroxyvitamin D3-24-hydroxylase (CYP24A1), 181 Hypercalcemia, 197

I Ibis T5000 platforms, 254 Ibuprofen, 152 Icterus, 175 iEESI. See Internal extractive electrospray ionization (iEESI) IHC images. See Immunohistochemical (IHC) images

Index

Imaging mass spectrometry (IMS), 267 Imipramine, broad spectrum drug screening, 127 Immunoaffinity extraction, 39 Immunoassays, 17, 18, 20 method, 251 platform manufacturers, 20 Immunohistochemical (IHC) images, 268 Immunometric reagents, 19 Immunosorbents, 23 Immunosuppressive agents, 166 cyclosporin A (CsA), 166 sirolimus, 166 tacrolimus, 166 IMS. See Imaging mass spectrometry (IMS) Incurred sample reanalysis (ISR), 67 Institutional buying, 168 Instrumentation, 213. See also Instrument maintenance; Instrument selection chromatography, 213 gas, 213 liquid, 213 mass spectrometry, 214 gas chromatography and, 214 liquid chromatography and, 214 Instrument maintenance, 100 liquid chromatography system, 101–102 autosampler maintenance, 102 maintenance of columns and guard columns, 101 replacement of LC tubing, 101 mass spectrometer maintenance, 102–103 cleaning, source and interface region, 102 resolution application and platform dependent, 103 Instrument manufacturers, 20 Instrument selection, 133 concentrations of analytes, 133 maintenance, 155 daily, 155 monthly, 156 weekly, 156 manufacturer considerations, 134 model, 134 periodic maintenance, 157 column maintenance, 157 filament, 158 ion source and ion volume, 157 pump oil, 157 troubleshooting, 155 Internal extractive electrospray ionization (iEESI), 267 Internal standards, 141 in mass spectrometry, 21 International Bureau of Weights and Measures (BIPM), 205 Internet jacks, 168 Intramolecular interactions, 39

281

Ion chromatograms, 252 Ion cyclotron resonance (ICR), 249 traps, 5 Ionic strength, 22 Ionization efficiencies, 21 Ionization source, 21 Ionization techniques choice of, 26–28 clinical mass spectrometry, 3 Ion mobility mass spectrometry, 268 Ion-pairing agents, 22 Ion suppression, 220 Ion trap (IT), 134–135, 262 Ion trap mass analyzers, 5 Isobaric isomers, 24 Isopropanol, 57 IT. See Ion trap (IT)

J Joint Committee Traceability in Laboratory Medicine (JCTLM), 205

K Karl-Fischer analysis, 85 Kidney Disease Outcomes Quality Initiative (KDOQI) guidelines, 196

L Laboratory-developed tests (LDTs), 63, 72, 167, 261 proposed new Federal regulations for, 72 Laboratory information system (LIS), 168 Lacosamide, 167, 169 LAESI. See Laser ablation electrospray ionization (LAESI) Lamotrigine, 167 Laser ablation electrospray ionization (LAESI), 267 LC-MS/MS. See Liquid chromatography mass spectrometry (LC-MS/MS); Liquid chromatography tandem mass spectrometry (LC-MS/MS) LC-MS/MS instruments, 17, 37. See also Liquid chromatography mass spectrometry (LC-MS/MS) LC-MS/MS sample preparation, 37 LC-MS technique, 20 analytical disadvantage of, 22 LC-QTOF for broad spectrum drug screening, 128–129 LC software, 104 LDTs. See Laboratory-developed tests (LDTs) Leflunomide, 167 Levetiracetam, 167 PT result, therapeutic drugs proficiency testing scheme, 177 Limit of detection (LOD), 175 Limit of quantitation (LOQ), 175

282

Index

Linearity, 66 Lipemia, 21, 175 Lipids, 23 Liquid chromatography mass spectrometry (LC-MS/MS), 183. See also Liquid chromatography (LC)-MS technology for toxicology testing, 111 advantages and disadvantages of, 111–112 high resolution mass spectrometry, 115–116 single quadrupole analyzers, 112 tandem mass spectrometry, 113–115 time-of-flight analyzers, 113 types of mass analyzers, 112 Liquid chromatography (LC)-MS technology, 1 challenges, 8 clinical applications, 6 endocrinology for, quantification of compounds, 6 external quality assessment and quality control, 8–9 key components of standard operating procedures (SOPs) for, 13 matrix effects and interferences, 9–11 need for, 6–7 potential pitfalls, 8 screening and confirmation of genetic disorders, 6 technical hurdles, 11–12 Liquid chromatography-tandem mass spectrometry (LC-MS/MS), 166, 214 field asymmetric ion mobility spectrometry (FAIMS), 216 high resolution mass spectrometry (HR-MS), 217 internal standard, 171 ionization, 215 ionization efficiency, 171 mass transition, 214 MS parameters, 216 cone /curtain gas, 216 dwell time, 216 source temperature, 216 sensitivity, 215 transitions for selected reaction, of nonderivatized steroid hormones, 212 Liquid EDLI. See Liquid electrospray laser desorption/ ionization (Liquid EDLI) Liquid electrospray laser desorption/ ionization (Liquid EDLI), 269 Liquid-liquid extraction (LLE), 10, 23, 142, 171 sample preparation protocol, 40 advantages, 40 disadvantages, 40 optimizing, 57 workflow, 40 Liquid-liquid extraction techniques, 23 LIS. See Laboratory information system (LIS) Listeria monocytogenes, 241 LLE. See Liquid-liquid extraction (LLE)

LLOQ. See Lower limit of quantitation (LLOQ) LOD. See Limit of detection (LOD) LOQ. See Limit of quantitation (LOQ) Lower limit of detection (LOD) definition, 120 Lower limit of quantitation (LLOQ), 39, 65, 152 Lyophilized materials, 85

M Magnetic beads, 23 MALDI. See Matrix-assisted laser desorption ionization (MALDI) MALDI-TOF imaging, 267 MALDI-TOF MS. See Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) Manufacturers, 20. See also Instrumentation Mass accuracy, definition, 1 Mass analyzer choice of, 29–32 Mass defect, 248 Mass spectrometric immunoassay (MSIA), 268 Mass spectrometry (MS), 17, 111, 165, 167, 168, 170, 231 in clinical diagnosis, 17 platform components, 20 platforms, 20 workflow, key steps in, 20 Mass spectrometry assay, steroid hormones sample tube types, choice of, 225 stability and storage, 225 validation, 217 accuracy, 217 carryover, 219 evaluation of linearity, 219 imprecision, 218 interferences, 220 ion suppression, 220 matrix effects, 220 sensitivity, 218–219 Mass spectrometry imaging (MSI), 255, 267 Mass-tagging approach, 192 Mass-to-charge ratio, 248 Matrix-assisted laser desorption ionization (MALDI), 28, 247 Matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF), 28, 253, 263 applications, 263 human herpesvirus detection, 263 oligonucleotide analysis, 263 microbial identification analyzers, 254 bioMérieux Vitek MS, 254 Bruker Biotyper, 254 Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), 231

Index

analysis, 231 working, 231 Matrix effects, 21, 41, 44, 52, 55, 220 Medications therapeutic drug monitoring clinical value, 169 Membrane inlet mass spectrometer (MIMS), 266 Meningitis, 236 Methodologies, limitations, including interfering substances, 78 Methotrexate, 167 Methylmalonic acid (MMA), 105 Microbiology, 253 laboratory, 235 MIMS. See Membrane inlet mass spectrometer (MIMS) Minimal sample preparation, 38 Mix-up pipetting, 171 Mobile phase, 13, 45, 82, 104, 126, 213. See also Stationary phase composition, 22 Molecular diagnostics, 254 Ibis T5000 platforms, 254 next generation sequencers (NGS), 254 polymerase chain reaction (PCR), 254 Sequenom MassARRAY, 254 Molecular imprinted polymers (MIPs), 39 Molecular weight (MW) range, 28 Monoisotopic mass, 248 Morphine, validation data for, 121, 122 MRM. See Multiple reaction monitoring (MRM) MS. See Mass spectrometry (MS) MSI. See Mass spectrometry imaging (MSI) MSIA. See Mass spectrometric immunoassay (MSIA) MS/MS interface, 56 MS/MS method, disadvantage of, 115 MTB. See M. tuberculosis complex (MTB) MTBE eluate, 58 M. tuberculosis complex (MTB), 238 Multiple-reaction monitoring (MRM), 4, 262 transitions, 19 Multiplex liquid chromatography system parallel LC Systems, 191 Mycobacterial liquid culture systems, 232 Mycobacteriology, 238 Mycology, 240 m/z gap, 21 m/z ratios, 29

N National Institute of Standards and Technology (NIST), 185 Neonatal intensive care unit, 240 Neutral or ion-pairing agents, 21 New assay implementation, 77 New born screening (NBS), 23

New materials, verification of, 83 Next generation sequencers (NGS), 254 NGS. See Next generation sequencers (NGS) Nicotine, 152 NIST. See National Institute of Standards and Technology (NIST) Nominal mass, 248 Nonextraction immunoassays, 188 Nonpolar wash solutions, 42 Nontuberculous mycobacteria (NTM), 238 Nonvolatile buffers, 22 Norclomipramine, broad spectrum drug screening, 127 Nordiazepam, thermal decomposition of, 147 Norfentanyl, validation data for, 121, 122 Normal subjects, association between 25(OH)D/24, 25(OH)2D, 198 NTM. See Nontuberculous mycobacteria (NTM) Nuclear translocation, 195

O 1,25(OH)2D assay procedure, 196 biochemistry and clinical utility, 195–196 1,25(OH)2D3 degradation rates, 195 metabolic clearance rate (MCR), 195 production rate (PR), 195 24,25(OH)2D assay procedure, 197 biochemistry and clinical utility, 196–197 24,25(OH)2D3 degradation rates, 195 metabolic clearance rate (MCR), 195 production rate (PR), 195 Online SPE, 43. See also Solid-phase extraction (SPE) additional LC pumps, 43 advantages of, 43 disadvantages, 43 Gerstel MPS, 43 optimizing, 58 risk for carryover, 43 Operational efficiency, 17 Opioid confirmation testing, 116, 118 chromatography conditions for, 119 determination of, lower limit of detection (LOD), 120 immunoassay value, 122 matrix effects, determinition, 120 method development, 119 considerations for, 117 rational for, 119 opioid metabolism, 116 postimplementation monitoring, 122 validation data, 120, 121

283

284

Index

Optimal chromatographic separation, 194 Orbitrap, 31, 262 Orbitrap Discovery mass spectrometer, 256 Oxazepam, thermal decomposition of, 147

P Pain management, 24 Pairing agents, 22 Paper spray (PS), 266 Parathyroid hormone (PTH), 181 Particulate-free filtrate, 40 Particulate free supernatant, 39 Parts per million (ppm), 249 Patient management, 165 PCR. See Polymerase chain reaction (PCR) PCR ESI-MS. See Polymerase chain reaction electrospray ionization mass spectrometry (PCR ESI-MS) PCR-MS, 264 Pentafluorobenzyl, as derivatizing agents used, in steroid hormone, 211 Peptidoglycan, 235 pH, 22, 54, 57, 58, 142 Phenotypic tests, 236 bile solubility, 236 optochin disk susceptibility, 236 Phenyl-1,2,4-triazoline-3,5-diones (PTAD), 192 Phosphate buffer, 22 Phospholipid removal plates, 23 Phospholipid removal sample preparation protocol (PLR), 41 media, 41 Phospholipids, 23, 40, 142, 189, 266 Photo isomerization, 181 Picolinoyl, as derivatizing agents used, in steroid hormone, 211 Pipette tips, 21 Polar analytes, 58 Polymerase chain reaction (PCR), 254 Polymerase chain reaction electrospray ionization mass spectrometry (PCR ESI-MS), 241 Postanalytical considerations, 225 reference ranges, 225 units and reporting, 226 Postimplementation monitoring, 222 data review, 222 external quality assessment, 222 harmonization, 222 proficiency testing, 222 Standardization of laboratory test, 222 ppm. See Parts per million (ppm) Preanalytical considerations, 223, 224 age and gender of patient, 223 sample type, 223 time of day of, sample collection, 223, 224

Precipitating agents, 39, 40 Precision, 48, 52, 65, 80, 117, 152, 263 longitudinal, 87 Precursor ions, 113 Process improvement, 20 Processing, and rejection criteria requirements, 78 Product ion (PI), 113 scan, 114 Proficiency testing (PT), 71, 173 Protein precipitation (PPT), 37, 171 sample preparation protocol, 39 advantages, 40 disadvantages, 40 optimizing, 55, 57 recommendation for ratio of precipitation reagent to serum, 56 Proteomics, 268 Protonated gas ions, 253 Proton transfer reaction (PTR), 265 PS. See Paper spray (PS) PTAD. See Phenyl-1,2,4-triazoline-3,5-diones (PTAD) PTH. See Parathyroid hormone (PTH) PTR. See Proton transfer reaction (PTR) Purification, analyte for detection by mass spectrometry, 17 Pyridyl, as derivatizing agents used, in steroid hormone, 211

Q qTOF. See Quadrupole-Time of Flight (qTOF) Quadrupole mass analyzers, 4 Quadrupole mass filter, 135, 136 Quadrupole-Time of Flight (qTOF), 262 Quality assurance, for specimen analysis, 17, 103–105, 176–177 cross-stream carryover, 105 leak checks and detection, 103 loss of mobile phase, 104 maintaining operating conditions, 105 MS platforms require pressurized gases for operation, 104 orientation of 96-well plates, 104 power supply to MS equipment, 105 pressure trace monitoring/ air bubbles, 104 sensitivity of mass spectrometry system, 104 Quality control (QC), 63, 87–89, 176–177 elements of, 70 proficiency testing (PT), 176 system suitability samples, 177 Quantitative toxicology, 170

R Radio immunoassays (RIAs), 181 Radiolabeled tracer, 184 Randomization, 90

Index

Raoultella ornitholyica, 235 Rapid evaporative ionization mass spectrometry (REIMS), 256, 268 Reagents, preparation of, 79 Real time analysis, 265 Recovery, 66 Reference intervals, 66 Reference material, 173 Regulatory compliance, 70 REIMS. See Rapid evaporative ionization mass spectrometry (REIMS) Relative retention times (RRT), 141 Reportable range, 66 Research use only (RUO) kits, 20 library, 234 Return on investment (ROI), 168 Reverse-phase chromatography (RPC), 119 Reverse-phase component, 42 RIAs. See Radio immunoassays (RIAs) Rickets, 181 Right mass spectrometer, 18 Right to use (RTU), 170 RLH. See Robotic liquid handlers (RLH) Robotic liquid handlers (RLH), 171 ROI. See Return on investment (ROI) Royal Society of Chemistry (RSC) documents concerning method development and validation, 72 RTU. See Right to use (RTU) RUO. See Research use only (RUO)

S Sample analysis, 89–90 Sample collection, 265 Sample identification, 171 Sample introduction, methods of, 138 direct probe analysis, 139 headspace injection, 139 solid phase microextraction (SPME), 139 solvent injection, 138 Sample preparation, 17, 19, 37, 142, 146, 265 analyte extraction, 142 considerations, 170–171 manual vs. automation, 171 sample type, 170–171 dispersive liquid-liquid microextraction (DLLME), 144 hydrolysis, 145, 146 for LCMS, 23 liquid-liquid extraction (LLE), 142, 171 protein crash, 171

285

protocol, 18. See also Sample preparation protocols selecting, 37 solid phase extraction, 142, 143 solid-phase extraction (SPE), 171 solid phase microextraction, 145 steps, 23 supported liquid extraction (SLE), 144 Sample preparation protocols, 38 comparison of, 50 assessing the feasibility of simple sample preparation, 51 cross-contamination, 52 DIL or PPT sample preparation, 52 LC-MS/MS sample preparation types, 50 low cost investment, 52 pH manipulation, 51 pH shift, 51 recovery for highly polar or permanently charged analytes, 51 retention, 51 selectivity, concentrating capability, and matrix removal, 51 sensitivity of the MS/MS, 51 throughput, precision, error rates, and labor productivity, 52 evaluation of, 44 evaluating chromatography, 44 evaluating matrix effect (ME), 44–45 evaluating signal to noise (S/N) at the LLOQ, 44 matrix mixing experiment, 48 phospholipid direct detection experiment, 46 qualitative postcolumn matrix effect experiment, 46 quantitative matrix effect experiment, 45 low cost (liquid-liquid extraction or LLE), 39 optimization of, 53, 57 generic-optimizing for 96-well format, 53 generic-optimizing LC injection solvent composition, 53 analyte solubility, 53 LC peak shape and Rt, 54 phospholipid removal media or PLR, 39 simple and low cost (DIL and PPT), 39 solid phase extraction or SPE, 39 supported liquid extraction or SLE, 39 SARAMIS. See Spectral archiving and microbial identification system (SARAMIS) Security-relevant library, 237 SELDI-TOF MS. See Surface-enhanced laser desorption/ ionization-Time of Flight (SELDI-TOF) MS Selected ion flow tube (SIFT), 265 Selected-reaction monitoring (SRM), 4, 114 Selectivity, 66 Send-out costs, 170 Sensitivity of the detector, 19 Sequenom MassARRAY, 254

286

Index

Serum gel separator tubes, 170 SIFT. See Selected ion flow tube (SIFT) Signature molecules, 18 Single droplet microextraction, 266 Single Quad GC-MS, 261 Single quadrupole analyzers, 112, 135–136 Single reaction monitoring (SRM), 30 transition, 30 Single stage high-resolution (HR) mass analyzers, 28 SISCAPA. See Stable isotope standards and capture by anti-peptide antibodies (SISCAPA) Skills, 19 Sodium periodate treatment, 186 Solid agar media, 232 Solid-phase extraction (SPE), 23, 142, 143, 171 sample preparation protocol, 42 advantages of, 43 bed require conditioning, 42 disadvantages, 43 eluate, 43 media with a polymer, 42 optimizing, 54 elution, 55 evaporation, 55 loading, 54 reconstitution, 55 stationary phase, 54 washing, 54 plates, 42 Solid phase microextraction (SPME), 139 Solutions, preparation of, 79 Solvent injection, 138 SPE. See Solid-phase extraction (SPE) Specimen collection, criteria requirements, 78 Specimen storage, and preservation, 79 Spectral archiving and microbial identification system (SARAMIS), 234 Split injection mode, 140 Splitless injection mode, 140 Sprectrophotometric detector, 22 16S rRNA sequencing, 237 Stability, of specimens, 67 Stable isotope standards and capture by anti-peptide antibodies (SISCAPA), 263 Standard calibration curve, 154 Standardization, 17 Standard operating procedure (SOP), 77, 175 adopted from the Clinical Laboratory Improvement Amendment, 77 Stationary phase, 24, 37, 54, 213 chemistry, 22 choice of, 24–25 STAT testing, 89 Step-by-step performance of the procedure, 79

Steroid hormone, clinical use, 205 calibration and quality control requirements, 205 purity of the material, 206 quality control materials, 210 standard materials, 205 standards preparation, 206 storage of crystalline materials, 206 classes of, 205 concentration range, 208 cyclopentanophenophenanthrene ring system, 205 instrumentation, 213 chromatography, 213 mass spectrometry, 214 internal standard, 209 selection of concentration of, 209 use of, 209 measurement, by mass spectrometry, 205 pathway of, 205 postanalytical considerations reference ranges, 225 units and reporting, 226 preanalytical considerations, 223, 224 age and gender of patient, 223 sample type, 223 time of day of, sample collection, 223, 224 sample preparation, 210 chemical derivatization, 211 derivatizing agents, 211 equilibrium dialysis, 210 extraction procedures, 211 isolation of analyte, 211 manipulation of, 210 preparation technique, choice of, 211 ultracentrifugation, 210 transitions for selected reaction, nonderivatized, 212 use of volatile solvents, 208 Steroid panel, 20 Sterols, 24 Strain-typing analysis, 240 Streptococcus mitis, 236 Streptococcus pyogenes, 266 Suboptimal chromatographic conditions, 21 Supported liquid extraction sample preparation protocol (SLE OR SALL), 41, 144 advantages of, 42 dilution of sample, 41 disadvantages, 42 Surface-enhanced laser desorption/ ionization-time of flight (SELDI-TOF) MS, 241 Synthetic cannabinoids, 251 System suitability test (SST) solutions, 80–83 basic definition for, 81 choice of solvent, 81 concentration of an operational, 81

Index

evaluation of the long-term stability, 81 frequency of SST analysis should be determined by, 82 isobaric pairs warranting inclusion in system suitability test solutions, 83 number of SST injections, 83

T Tacrolimus imprecision data by LC-MS/MS, 172 linearity over twenty runs by LC-MS/MS, 166 Tandem mass spectrometry, 23, 113–115 for broad spectrum drug screening, 113 in clinical toxicology laboratories, 114 for drug confirmatory testing, 113 MS scan modes, 114 Tandem quadrupole mass spectrometer, 4 TAT. See Turn-around-time (TAT) TDM. See Therapeutic drug monitoring (TDM) Technical assistance, 20 Temazepam, thermal decomposition of, 147 Temperature, 10, 22, 55, 69, 79, 104, 128, 140, 157, 208, 225 Test matrix, 170 Testosterone, 20, 249 mass resolution, 250 TFA, ion-pairing agents, 22 Therapeutic drug monitoring (TDM), 165, 167, 169, 262 applications internal standard choice, 171 MS considerations, 165–169 clinical need/utility, 165–167 financial considerations, 168 physical considerations, 168 strategic considerations, 169 technical considerations, 167 Therapeutic medications, MS monitoring, by, 166 Therapeutic substance monitoring, 170 Thin layer chromatography, 110 TIGER. See Triangulation identification for the genetic evaluation of risk (TIGER) Time of flight (TOF) analyzers, 4, 28, 113, 137, 233 fast scan rates, 137 sensitivities, 137 TM. See Transmission mode (TM) TOF. See Time of flight (TOF) Total testosterone analysis clot activator tube vs serum separator tube, 221 Touch spray (TS), 266 Toxicology, 24 laboratory, 251 Toxicology testing, clinical applications, 116 opioid confirmation testing, 116

287

chromatographic methods for, 110 gas chromatography mass spectrometry, 111 liquid chromatography mass spectrometry, 111 thin layer chromatography, 110 definition, 109 diagnosis and treatment, 110 ToxTyper software, 262 Traditional biochemical methods, 235 Training, 19 Transmission mode (TM), 266 Triangulation identification for the genetic evaluation of risk (TIGER), 254 Trichloroacetic acid (TCA), 56 Triple quad. See Triple quadrupole (Triple quad) Triple Quad GC-MS, 261 Triple Quad LC-MS, 262 Triple quadrupole. See Tandem quadrupole mass spectrometer Triple quadrupole (Triple quad), 136–137, 261 Single/Triple Quad GC-MS, 261 Triple Quad LC-MS, 262 Triple quadrupole mass spectrometry, 19 Troubleshooting, 158 determination of chromatographic issues, 158 mold-release agents, 159 preanalytical factors, 158 sources of contamination, 158 TS. See Touch spray (TS) TurboFlow technology, 52, 189 Turn-around-time (TAT), 165

U Ultra-pressure liquid chromatography (UPLC), 197 United States, National Institute of Standards and Technology (NIST), 205 UPLC. See Ultra-pressure liquid chromatography (UPLC) Upper limit of quantitation (ULOQ), 65 US Food and Drug Administration (FDA) Guidance, 20, 65, 165 for Industry on bioanalytical method validation, 64

V Vacuum flight tube, 232 Validation considerations, 172–176 accuracy, 173 additional validation studies, 176 carry-over, 175 imprecision, 172 ion suppression/enhancement, 175 reference intervals, 173 reportable range, 173 sensitivity, 175 specificity (interferences), 175

288

Index

Vaporization chamber, 23 VBP. See Vitamin D, binding protein (VBP) VDBG. See Vitamin D, binding globulin (VDBG) VDDR I. See Vitamin D, dependent rickets type I (VDDR I) VDDR II. See Vitamin D, dependent rickets type II (VDDR II) Verification. See also Validation of performance characteristics, 66 Vitamin D binding globulin (VDBG), 262 binding protein (VBP), 181 dependent rickets type I (VDDR I), 196 dependent rickets type II (VDDR II), 196 external quality assessment scheme (DEQAS), 185 2016 report, 186 metabolic pathway, 182 metabolite quantitation, 183 biochemical effect on PTH, 184 biochemical effect on serum calcium, 184 clinical utility, 184 metabolites evolution of assays, 183–186 LC-MS/MS quantitation, 188–197 assay performance parameters, 188 sample preparation steps, 190 physiological role, 181

toxicity, 197 Vitamin D2 assay procedure, 189 clinical utility, 188 Vitamin D3 absorbtion, 188 via intestinal lymphatics, 188 Vitamin D3 assay procedure, 189 clinical utility, 188 VOCs. See Volatile organic compounds (VOCs) Volatile analytes, 24 Volatile organic compounds (VOCs), 265 Volatile sampling, 265 Voraxaze, 167 Vortex mixing, 171

W Workflows, 11, 20, 21, 29, 46, 231, 232, 264 commercialized as PLEX-ID, 254 LC-MS, 11 MALDI-TOF MS, 232 pre- and postanalytical, 11

Y Yersinia pestis, 237